Glycoengineered antibody drug conjugates

ABSTRACT

The current disclosure provides binding polypeptides (e.g., antibodies), and targeting moiety conjugates thereof, comprising a site-specifically engineered glycan linkage within native or engineered glycans of the binding polypeptide. The current disclosure also provides nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Methods of using the antigen-binding polypeptides disclosed herein to treat disease are also provided.

RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalPatent Application No. 62/061,989, filed Oct. 9, 2014. The entirecontent of the aforementioned application is incorporated by referencein its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 17, 2015, isnamed 571591SA9-166_SL.txt and is 114,775 bytes in size.

BACKGROUND

Use of specific antibodies to treat people and other animals is apowerful tool that has been very effective in treating many conditionsand disorders. However, there is great demand for more effectivetargeted therapeutics, especially target specific therapies with higherefficacy and greater therapeutic window. One of these target specifictreatments employs antibody-effector moiety conjugates in which atargeting moiety directs a specific antibody to a desired treatmentsite. These molecules have shown improved therapeutic index—higherefficacy and/or lower toxicity profiles than the un-targeted antibody ina clinical setting. However, development of such therapeutics can bechallenging as many factors, including the antibody itself and linkagestability, can have significant impact on the disease target (e.g.tumor) specificity, thereby reducing efficacy. With high non-specificbinding and low stability in circulation, the antibody-effector moietyconjugate would be cleared through normal tissues before reaching thetarget site. Moreover, antibody-effector moiety conjugates withsignificant subpopulations of high drug loading could generateaggregates which would be eliminated by macrophages, leading to shorterhalf-life. Thus, there are increasing needs for critical process controland improvement as well as preventing complications such as the productaggregation and nonspecific toxicity from antibodies.

Although antibody-effector moiety conjugates generated according tocurrent methods are effective, development of such therapeutics can bechallenging as heterogeneous mixtures are often a consequence of theconjugation chemistries used. For example, effector moiety conjugationto antibody lysine residues is complicated by the fact that there aremany lysine residues (˜30) in an antibody available for conjugation.Since the optimal number of conjugated effector moiety to antibody ratio(DAR) is much lower to minimize loss of function of the antibody (e.g.,around 4:1), lysine conjugation often generates a very heterogeneousprofile. Furthermore, many lysines are located in critical antigenbinding sites of CDR region and drug conjugation may lead to a reductionin antibody affinity. On the other hand, while thiol mediatedconjugation mainly targets the eight cysteines involved in hingedisulfide bonds, it is still difficult to predict and identify whichfour of eight cysteines are consistently conjugated among the differentpreparations. More recently, genetic engineering of free cysteineresidues has enabled site-specific conjugation with thiol-basedchemistries, but such linkages often exhibit highly variable stability,with the linker undergoing exchange reactions with albumin and otherthiol-containing serum molecules. Finally, oxidizing agents (such asperiodate oxidase and galactose oxidase) used to treat antibodies inpreviously developed conjugation protocols can cause over-oxidation andextraneous oxidation of the binding polypeptide, reducing efficiency andefficacy of the conjugation itself.

Therefore, a site-specific conjugation strategy which generates anantibody conjugate with a defined conjugation site and stable linkagewithout the use of oxidizing agents would be highly useful inguaranteeing effector moiety conjugation while minimizing adverseeffects on antibody structure or function.

SUMMARY

The current disclosure provides methods of making effector moietyconjugates (e.g., targeting moiety conjugates). These methods involvethe incorporation of sialic acid derivatives in the glycan of a bindingpolypeptide to form a sialic acid derivative-conjugated bindingpolypeptide, and a subsequent reaction in which an effector moiety isreacted with the sialic acid derivative-conjugated binding protein tocreate an effector moiety-conjugated binding polypeptide.

In one aspect, the instant disclosure provides methods of making aneffector moiety conjugated binding polypeptide comprising the steps of:(a) reacting a cytidine monophosphate-sialic acid (CMP-sialic acid)derivative with a glycan of a binding polypeptide to form a sialic acidderivative-conjugated binding polypeptide; and (b) reacting the sialicacid derivative-conjugated binding polypeptide with an effector moietyto form the effector moiety conjugated binding polypeptide, wherein animine bond is formed, and wherein neither the binding polypeptide northe sialic acid derivative-conjugated binding polypeptide are treatedwith an oxidizing agent.

In one embodiment, the sialic acid derivative-conjugated bindingpolypeptide comprises a terminal keto or aldehyde moiety. In anotherembodiment, the effector moiety comprises a terminal aminooxy moiety oris bound to a moiety comprising an aminooxy derivative. In a furtherembodiment, the effector moiety is selected from those in FIGS. 45 and46.

In one embodiment, step (b) results in the formation of an oxime bond.In another embodiment, the effector moiety comprises a terminalhydrazine. In a specific embodiment, step (b) results in the formationof a hydrazone linkage. In a further embodiment, the effector moiety hasone or more of the following structural formulas:

In one aspect, the instant application provides methods of making aneffector moiety conjugated binding polypeptide comprising the steps of:(a) reacting a CMP-sialic acid derivative comprising a terminal reactivemoiety at the C5 position with a glycan of a binding polypeptide to forma sialic acid derivative-conjugated binding polypeptide; and (b)reacting the sialic acid derivative-conjugated binding polypeptide withan effector moiety to form the effector moiety conjugated bindingpolypeptide using click chemistry.

In one embodiment, the terminal reactive moiety is an azide, wherein theeffector moiety comprises an alkyne or is bound to a moiety comprisingan alkyne, and wherein step (b) forms a triazole ring at or linked tothe C5 position of the sialic acid derivative. In another embodiment,neither the binding polypeptide nor the sialic acidderivative-conjugated binding polypeptide are treated with an oxidizingagent. In another embodiment, the CMP-sialic acid derivative has astructural formula selected from the following:

In another embodiment, the effector moiety comprises or is bound to acyclooctyne. In a specific embodiment, the cyclooctyne is anazadibenzocyclooctyne. In another embodiment, step (b) occurs at ambienttemperatures. In another embodiment, step (b) is performed in theabsence of copper.In one aspect, the instant application provides methods of making aneffector moiety conjugated binding polypeptide comprising the steps of:(a) reacting a CMP-sialic acid derivative with a glycan of a bindingpolypeptide to form a sialic acid derivative-conjugated bindingpolypeptide; and (b) reacting the sialic acid derivative-conjugatedbinding polypeptide with an effector moiety to form the effector moietyconjugated binding polypeptide, wherein a thioether bond is formed.

In one embodiment, neither the binding polypeptide nor the sialic acidderivative-conjugated binding polypeptide are treated with an oxidizingagent. In another embodiment, the sialic acid derivative comprises aterminal thiol moiety. In another embodiment, the effector moietycomprises a maleimide moiety. In another embodiment, the effector moietyis bis-mannose-6-phosphate hexamannose maleimide, lactose maleimide, orany other component comprising at least one maleimide moiety of thefollowing structural formula:

In one embodiment, the effector moiety comprises one or more proteins,nucleic acids, lipids, carbohydrates, or combinations thereof. Inanother embodiment, the effector moiety comprises a glycan. In aspecific embodiment, the effector moiety comprises one or moreglycoproteins, glycopeptides, or glycolipids.

In another embodiment, the binding protein has one or more native orengineered glycosylation sites. In a further embodiment, the methodcomprising achieving or modifying the glycosylation of the bindingprotein using one or more glycosyltransferases, one or moreglycosidases, or a combination thereof. In another embodiment, step (a)occurs in a reaction with sialyltransferase. In a further embodiment,the sialyltransferase is a mammalian sialyltransferase. In a specificembodiment, the sialyltransferase is beta-galactosidealpha-2,6-sialyltransferase 1. In one embodiment, the effector moietybinds to a cell. In a further embodiment, the cell is selected from animmune cell, a liver cell, a tumor cell, a vascular cell, an epithelialcell, or a mesenchymal cell. In another embodiment, the cell is selectedfrom a B cell, a T cell, a dendritic cell, a natural killer (NK) cell, amacrophage, a hepatocyte, a liver sinusoidal endothelial cell, or ahepatoma cell.

In one embodiment, the effector moiety binds to a mannose 6 phosphatereceptor on the cell. In a further embodiment, the effector moietycomprises a mannose 6 phosphate moiety. In another embodiment, theeffector moiety binds to a Siglec on the cell. In a further embodiment,the Siglec is sialoadhesin (Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3),MAG (Siglec-4), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9,Siglec-10, Siglec-11, Siglec-12, Siglec-14, or Siglec-15. In anotherembodiment, the effector moiety binds to a C-type lectin receptor, agalectin, or an L-type lectin receptor on the cell. In a furtherembodiment, the effector moiety binds to TDEC-205, macrophage mannosereceptor (MMR), Dectin-1, Dectin-2, macrophage-inducible C-type lectin(Mincle), dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN,CD209), DC NK lectin group receptor-1 (DNGR-1), Langerin (CD207), CD169,a lectican, an asialoglycoprotein receptor (ASGPR), DCIR, MGL, a DCreceptor, a collectin, a selectin, an NK-cell receptor, a multi-CTLDendocytic receptor, a Reg group (type VII) lectin, chondrolectin,tetranectin, polycystin, attractin (ATRN), eosinophil major basicprotein (EMBP), DGCR2, Thrombomodulin, Bimlec, SEEC, orCBCP/Frem1/QBRICK.

In one embodiment, the effector moiety is a glycopeptide capable ofbinding ASGPR on a cell. In a further embodiment, the effector moiety isa trivalent GalNAc glycan containing glycopeptide or a trivalentgalactose containing glycopeptide. In a specific embodiment, theeffector moiety is represented by Formula V:

In another specific embodiment, the effector moiety is represented byFormula VI:

In one embodiment, the binding polypeptide comprises an Fc domain. Inanother embodiment, a modified glycan is N-linked to the bindingpolypeptide via an asparagine residue at amino acid position 297 of theFc domain, according to EU numbering. In another embodiment a modifiedglycan is N-linked to the binding polypeptide via an asparagine residueat amino acid position 298 of the Fc domain, according to EU numbering.In a further embodiment, the Fc domain is human.

In another embodiment, the binding polypeptide comprises a CH1 domain.In a further embodiment, a modified glycan is N-linked to the bindingpolypeptide via an asparagine residue at amino acid position 114 of theCH1 domain, according to Kabat numbering.

In a specific embodiment, the binding polypeptide is an antibody orimmunoadhesin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the formation of exemplaryCMP-sialic acid derivatives from sugar or sugar derivatives.

FIG. 2 is a schematic illustration of exemplary CMP-sialic acidderivatives.

FIG. 3 is a series (A-E) of depictions of different chemical reactionsof the instant invention, the circles in combination with the reactivemoieties to which they are bonded represent sialic acidderivative-conjugated binding polypeptides. The stars representtargeting or effector moieties.

FIG. 4 depicts an example of an effector moiety-conjugated bindingpolypeptide according to the methods illustrated in FIG. 3 (parts A-C)with a sialic acid derivative shown in FIG. 2.

FIG. 5 depicts an example of an effector moiety-conjugated bindingpolypeptide according to the methods illustrated in FIG. 3D with asialic acid derivative shown in FIG. 2.

FIG. 6 is a schematic illustration of the synthesis of an antibody drugconjugate where a toxin moiety is linked to an oxidized sialic acidresidue of the antibody glycan using an oxime linkage.

FIG. 7 is a Coomassie-blue stained gel showing the expression andpurification of glycosylation mutants.

FIG. 8 depicts the results of surface plasmon resonance experiments usedto assess the binding of αβTCR HEBE1 IgG antibody mutants to recombinanthuman FcγRIIIa (V158 & F158).

FIG. 9 depicts the results of surface plasmon resonance experiments usedto assess the binding of αβTCR HEBE1 IgG antibody mutants to recombinanthuman FcγRI.

FIG. 10 depicts the cytokine release profile from PBMCs for TNFa,GM-CSF, IFNy and IL10 in the presence of mutant anti-αβTCR antibodies(day 2).

FIG. 11 depicts the cytokine release profile from PBMCs for IL6, IL4 andIL2 in the presence of mutant anti-αβTCR antibodies (day 2).

FIG. 12 depicts the cytokine release profile from PBMCs for TNFa,GM-CSF, IFNy and IL10 in the presence of mutant anti-αβTCR antibodies(day 4).

FIG. 13 depicts the cytokine release profile from PBMCs for IL6, IL4 andIL2 in the presence of mutant anti-αβTCR antibodies (day 4).

FIG. 14 depicts the results of experiments investigating the expressionlevel of 2C3 mutants by Western blotting (A) and surface plasmonresonance (B).

FIG. 15 depicts the results of experiments investigating glycosylationof 2C3 mutants pre- and post-PNGase F treatment.

FIG. 16 depicts the results of SDS-PAGE experiments investigatingglycosylation sites on 2C3 mutants isolated from cell culture.

FIG. 17 depicts the results of surface plasmon resonance experimentsused to assess the binding of modified anti-CD52 to recombinant humanFcγRIIIa (V158). Anti-CD52 comprising S298N/Y300S mutations in the Fcdomain were used to assess the effector function of the modifiedmolecule. binding to CD52 peptide (A), binding to FcγRIIIa (V158, B),and control binding to mouse FcRn (C).

FIG. 18 depicts the results of surface plasmon resonance experimentsinvestigating the Fc binding properties of 2C3 mutants.

FIG. 19 depicts the results of surface plasmon resonance experimentsinvestigating the binding of modified anti-CD52 to both FcγRIIIa(Val158) (as above) and FcγRIIIa (Phe158). Anti-CD52 antibodiescomprising S298N/Y300S mutations in the Fc domain were used to assessthe effector function of the modified molecule binding to FcγRIIIa(Val158, A) and FcγRIIIa (Phe58, B).

FIG. 20 depicts the analysis of C1q binding in the S298N/Y300S mutantand the WT 2C3 control (A) and the results of an Eliza analysisconfirming equivalent coating of the wells (B).

FIG. 21 depicts the results of plasmon resonance experiments measuringthe binding kinetics of 2C3 mutants to CD-52 peptide 741.

FIG. 22 depicts the results of plasmon resonance experiments comparingthe antigen binding affinity of WT anti-CD-52 2C3 and the A114Nhyperglycosylation mutant.

FIG. 23 depicts the results of isoelectric focusing and massspectrometry charge characterization experiments (A-D) to determine theglycan content of 2C3 mutants.

FIG. 24 depicts the results of concentration (A; Octet) and plasmonresonance experiments (B) comparing the antigen binding affinity of WTanti-CD52 2C3 and mutants.

FIG. 25 depicts the results of SDS-PAGE experiments to demonstrate theadditional glycosylation of the anti-TEM1 A114N mutant.

FIG. 26 depicts the results of SDS-PAGE and hydrophobic interactionchromatography analysis of the A114N anti-Her2 mutant.

FIG. 27 depicts the results of SDS-PAGE experiments to demonstrate theconjugation of PEG to the 2C3 A114N mutant through an aminooxy linkage.

FIG. 28 depicts the results of LC-MS experiments to determine the glycancontents of anti-TEM1 A114N hyperglycosylation mutant.

FIG. 29 depicts the results of LC-MS experiments to determine the glycancontents of a wild-type HER2 antibody and an A114N anti-Her2hyperglycosylation mutant.

FIG. 30 depicts an alternative method for performing site-specificconjugation of an antibody comprising the use of oxidizing agents (A-C).

FIG. 31 depicts a synthesis of exemplary effector moieties:aminooxy-Cys-MC-VC-PABC-MMAE and aminooxy-Cys-MC-VC-PABC-PEGS-Dol10.

FIG. 32 depicts characterization information (A-C) for a sialylated HER2antibody.

FIG. 33 depicts characterization information (A-D) for oxidizedsialylated anti-HER 2 antibody.

FIG. 34 depicts hydrophobic interaction chromatographs ofglycoconjugates prepared with three different sialylated antibodies withtwo different aminooxy groups.

FIG. 35 shows a HIC chromatograph of anti-Her2 A114 glycosylation mutantconjugate with AO-MMAE prepared using GAM(+) chemistry.

FIG. 36 depicts a comparison of the in vitro potency of an anti-HER2glycoconjugate and thiol conjugate (A-D).

FIG. 37 depicts a comparison of the in vitro potency of an anti FAP B11glycoconjugate and thiol conjugate.

FIG. 38 depicts a comparison of in vivo efficacy of anti-HER2glycoconjugates and thiol conjugates in a Her2+ tumor cell xenograftmodel (A-D).

FIG. 39 depicts the results of LC-MS experiments to determine the glycancontent of a mutant anti-αβTCR antibody containing the S298N/Y300Smutation. FIG. 39 discloses “NNAS” as SEQ ID NO: 40.

FIG. 40 depicts the results of circular dichroism experiments todetermine the relative thermal stability of a wild-type anti-αβTCRantibody and mutant anti-αβTCR antibody containing the S298N/Y300Smutation.

FIG. 41 depicts the results of a cell proliferation assay for ADCprepared with the anti-HER antibody bearing the A114N hyperglycosylationmutation and AO-MMAE.

FIG. 42 is a schematic illustration of an alternative synthesis of anantibody drug conjugate where a targeting moiety is linked to anoxidized sialic acid residue of the antibody glycan using an oximelinkage. This alternative synthesis makes use of oxidizing agents.

FIG. 43 is a schematic illustration depicting an alternative method forperforming site-specific conjugation of an antibody to a glycopeptidethrough an aminooxy linkage according to the disclosed methods. Thisalternative synthesis makes use of oxidizing agents.

FIG. 44 is a schematic illustration depicting an alternative method ofsite-specific conjugation of neoglycans to antibody through sialic acidin native Fc glycans. This alternative synthesis makes use of oxidizingagents.

FIG. 45 is a series of exemplary glycans that may be used forconjugation including lactose aminooxy and bis M6P hexamannose aminooxy(for aminooxy conjugation).

FIG. 46 is a schematic depiction the preparation of M-6-P hexamannosemaleimide.

FIG. 47 depicts SDS-PAGE and MALDI-TOF characterization of Man-6-Phexamannose aminooxy conjugates made with rabbit polyclonal antibody.

FIG. 48 depicts the results of surface plasmon resonance experimentsused to assess the binding of control and Man-6-P hexamannose conjugatedrabbit IgG antibodies to M6P receptor.

FIG. 49 depicts the uptake of Man-6-P conjugated rabbit IgG antibody inHepG2 and RAW cells.

FIG. 50 depicts the characterization of control, Man-6-P conjugated, andlactose conjugated antibodies through SDS-PAGE and lectin blotting.

FIG. 51 depicts the results of MALDI-TOF intact protein analyses forcontrol, Man-6-P conjugated, and lactose conjugated antibodies.

FIG. 52 depicts the characterization of polyclonal antibody conjugatedto Man-6-P hexamannose maleimide (thiol conjugation at hinge cysteines)through SDS-PAGE (non-reducing and reducing), lectin blot (reducing),and M6P quantitation.

FIG. 53 depicts the characterization of polyclonal antibody conjugatedto lactose maleimide (thio conjugation at hinge cysteines) throughSDS-PAGE and galactose quantitation.

FIG. 54 depicts the characterization of monoclonal antibody conjugatedto Man-6-P hexamannose maleimide (thiol conjugation at hinge cysteines)through SDS-PAGE (non-reducing and reducing), and glycan (M6P)quantitation.

FIG. 55 depicts the results of size exclusion chromatography (SEC)analysis of a hinge cysteine polyclonal antibody conjugate.

FIG. 56 depicts the results of size exclusion chromatography (SEC)analysis of a hinge cysteine monoclonal antibody conjugate.

FIG. 57 depicts the results of sialidase titration to determine theamount of sialic acid release from NNAS (“NNAS” disclosed as SEQ ID NO:40), sialylated NNAS (“NNAS” disclosed as SEQ ID NO: 40), anddesialylated and galatosylated NNAS antibodies (“NNAS” disclosed as SEQID NO: 40). FIG. 57 discloses “NNAS” as SEQ ID NO: 40.

FIG. 58 depicts the results of LC-MS experiments to determine the glycancontents of an NNAS modified antibody (“NNAS” disclosed as SEQ ID NO:40) and a desialylated and galactosylated NNAS modified antibody (“NNAS”disclosed as SEQ ID NO: 40). FIG. 58 discloses “NNAS” as SEQ ID NO: 40.

FIG. 59 depicts the results of LC-MS experiments to determine the glycancontents of an NNAS modified antibody (“NNAS” disclosed as SEQ ID NO:40) and a sialylated NNAS modified antibody (“NNAS” disclosed as SEQ IDNO: 40). FIG. 59 discloses “NNAS” as SEQ ID NO: 40.

FIG. 60 depicts the characterization of M6P Receptor bound to bisM6Pglycan-conjugated polyclonal and monoclonal antibodies through native Fcglycan or hinge disulfides in solution.

FIG. 61 depicts the characterization of enzyme modified and conjugatedNNAS (“NNAS” disclosed as SEQ ID NO: 40) antibodies by SDS-PAGE (4-12%NuPAGE; reducing and non-reducing) and ECL lectin blotting (reducing).

FIG. 62 depicts the results of terminal galactose quantitation in anNNAS antibody (“NNAS” disclosed as SEQ ID NO: 40), adisialylated/galactosylated NNAS antibody (“NNAS” disclosed as SEQ IDNO: 40), and a conjugated NNAS antibody (“NNAS” disclosed as SEQ ID NO:40) in mol galactose or mol glycopeptide per mol antibody. FIG. 62discloses “NNAS” as SEQ ID NO: 40.

FIG. 63 depicts the examination of lactose maleimide that had beenmodified with alpha-2,3-sialyltransferase and eluted from QAEpurification columns with 20 mM NaCl. The resultant eluate wascharacterized using MALDI-TOF and Dionex HPLC.

FIG. 64 depicts the characterization of rabbit antibody conjugated withsialyllactose maleimide (thiol reaction) using SDS-PAGE (A) and DionexHPLC (B; sialic acid quantitation).

FIG. 65 depicts the characterization of lactose maleimide sialylatedwith alpha-2,6-sialyltransferase and purified using a QAE-sepharosecolumn. Analysis using Dionex HPLC is shown for (A) a lactose standard;(B) an alpha-2,6-sialyllactose standard; (C) a lactose maleimidestandard; and (D) a fraction of alpha-2,6-sialyllactose maleimide elutedfrom a QAE-sepharose column.

FIG. 66 depicts the characterization of a fraction ofalpha-2,6-sialyllactose maleimide eluted from a QAE-sepharose columnusing MALDI-TOF.

FIG. 67 depicts the characterization of a control antibody, analpha-2,3-sialyllactose glycan conjugated polyclonal antibody, and analpha-2,6-sialyllactose glycan conjugated polyclonal antibody throughSDS-PAGE (A) and Dionex HPLC (B; graph of sialic acid analysis shown).

FIG. 68 depicts the characterization of control and enzyme modified(disalylated/galactosylated) NNAS mutant antibodies (“NNAS” disclosed asSEQ ID NO: 40) using SDS-PAGE and lectin blotting.

FIG. 69 depicts the characterization through reducing SDS-PAGE of thePEGylation of a control antibody and Gal NNAS (“NNAS” disclosed as SEQID NO: 40) with various amounts of galactose oxidase. FIG. 69 discloses“NNAS” as SEQ ID NO: 40.

FIG. 70 depicts the results from a Protein Simple scan characterizingthe PEGylation of an antibody heavy chain.

FIG. 71 depicts the characterization through reducing SDS-PAGE of thePEGylation of a control antibody and Gal NNAS (“NNAS” disclosed as SEQID NO: 40) with various molar excess of PEG over antibody. FIG. 71discloses “NNAS” as SEQ ID NO: 40.

FIG. 72 depicts the results from a Protein Simple scan characterizingthe PEGylation of an antibody heavy chain.

FIG. 73 is a structural drawing of lactose₃-Cys₃Gly₄.

FIG. 74 depicts the characterization through reducing SDS-PAGE of thePEGylation of a control antibody and Gal NNAS (“NNAS” disclosed as SEQID NO: 40) with galactose oxidase in the absence of copper acetate (A)and in the presence of varying amounts of copper acetate (A and B). FIG.74 discloses “NNAS” as SEQ ID NO: 40.

FIG. 75 the characterization of enzyme modified and conjugated wildtype, A114N, NNAS (“NNAS” disclosed as SEQ ID NO: 40), and A114N/NNASantibodies (“NNAS” disclosed as SEQ ID NO: 40) by SDS-PAGE (4-12%NuPAGE; reducing and non-reducing) and ECL lectin blotting (reducing)along with the results of terminal galactose quantitation in molgalactose per mol antibody. FIG. 75 discloses “NNAS” as SEQ ID NO: 40.

FIG. 76 is a graph depicting the sialic acid content (in mol/mol) ofwild-type and mutant antibodies as measured using Dionex HPLC. FIG. 76discloses “NNAS” as SEQ ID NO: 40.

FIG. 77 depicts the characterization of the PEGylation of wild-type andmutant antibodies through reducing and non-reducing SDS-PAGE. FIG. 77discloses “NNAS” as SEQ ID NO: 40.

FIG. 78 is a graph depicting the PEGylation (in mol/mol) of wild-typeand mutant antibodies. FIG. 78 discloses “NNAS” as SEQ ID NO: 40.

FIG. 79 is a series of photos depicting immunofluorescence stainingresults from the incubation of control, modified (withgalactosyltransferase), or conjugated (with lactose aminooxy or lactosemaleimide) antibodies with HepG2 cells.

FIG. 80 is a depiction of a trivalent GalNAc glycan

FIG. 81 depicts the results of surface plasmon resonance experimentsused to assess the binding of trivalent GalNAc glycan-conjugatedantibodies to ASGPR receptor subunit H1.

FIG. 82 is a depiction of a trivalent GalNAc-containing glycopeptide anda trivalent galactose-containing glycopeptide.

FIG. 83 depicts the results of surface plasmon resonance experimentsused to assess the binding of trivalent GalNAc-conjugated and trivalentgalactose-conjugated recombinant lysosomal enzymes to ASGPR receptorsubunit H1.

FIG. 84 is a graph depicting the titration of sialic acid (0.2 μmol)with various amounts of CMP-sialic acid synthetase (N. mentingitidis) at37° C. as CMP-sialic acid synthesized versus the amounts of enzyme used.

FIG. 85 is a graph depicting the synthesized sialic acid (from ManNAc)versus the amounts of the sialic acid aldolase (E. coli K-12) enzymeused at 37° C.

FIG. 86 is a graph depicting the synthesized sialic acid derivative(from ManLev) versus the amounts of the sialic acid aldolase (E. coliK-12) enzyme used at 37° C.

FIG. 87 is a graph depicting the released sialic acid derivative afterdigestion of CMP-sialic acid derivative (synthesized from ManLev) withsialidase at 37° C. as compared to the retention time of sialic acidstandard monitored using HPAEC-PAD.

FIG. 88 is a graph depicting the HPAEC-PAD profile of CMP-sialic acidsynthesized from ManNAc and CMP-sialic acid derivative synthesized fromManLev as compared to the CMP-sialic acid standard.

FIG. 89 is a graph depicting the HPAEC-PAD profile of CMP-sialic acidderivatives (synthesized from ManLev, ManNAz and ManAz) as compared tothe CMP-sialic acid standard.

FIG. 90 is a schematic representation demonstrating the sialylation ofantibody using a CMP-sialic acid derivative prepared from ManLev.

FIG. 91 is a graph showing the LC-MS analysis of CH₂CH₃ fragmentsreleased by IdeS protease from antibody Herceptin sialylated in vitrousing α2,6 sialyltransferase and CMP-sialic acid derivative preparedfrom ManLev.

FIG. 92 is a schematic representation demonstrating the PEGylation ofantibody sialylated with a sialic acid derivative prepared from ManLev.

FIG. 93 depicts SDS-PAGE characterization of PEGylated Herceptinpre-sialylated with a sialic acid derivative prepared from ManLev. ThePEGylation is performed using oxime chemistry.

FIG. 94 is a schematic representation demonstrating the sialylation ofantibody using a CMP-sialic acid derivative prepared from ManNAz.

FIG. 95 depicts SDS-PAGE characterization of PEGylated Herceptinpre-sialylated with a sialic acid derivative prepared from ManNAz. ThePEGylation was performed using click chemistry.

DETAILED DESCRIPTION

The current disclosure provides methods of making effector moietyconjugates (e.g., targeting moiety conjugates). These methods involvethe incorporation of sialic acid derivatives in the glycan of a bindingpolypeptide to form a sialic acid derivative-conjugated bindingpolypeptide, and a subsequent reaction in which an effector moiety isreacted with the sialic acid derivative-conjugated binding protein tocreate an effector moiety-conjugated binding polypeptide.

I. Definitions

As used herein, the term “binding protein” or “binding polypeptide”shall refer to a polypeptide (e.g., an antibody) that contains at leastone binding site which is responsible for selectively binding to atarget antigen of interest (e.g. a human antigen). Exemplary bindingsites include an antibody variable domain, a ligand binding site of areceptor, or a receptor binding site of a ligand. In certain aspects,the binding polypeptides comprise multiple (e.g., two, three, four, ormore) binding sites. In certain aspects, the binding protein is not atherapeutic enzyme.

As used herein, the term “native residue” shall refer to an amino acidresidue that occurs naturally at a particular amino acid position of abinding polypeptide (e.g., an antibody or fragment thereof) and whichhas not been modified, introduced, or altered by the hand of man. Asused herein, the term “altered binding protein” or “altered bindingpolypeptide” includes binding polypeptides (e.g., an antibody orfragment thereof) comprising at least one non-native mutated amino acidresidue.

The term “specifically binds” as used herein, refers to the ability ofan antibody or an antigen-binding fragment thereof to bind to an antigenwith a dissociation constant (Kd) of at most about 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M, or less, and/or tobind to an antigen with an affinity that is at least two-fold greaterthan its affinity for a nonspecific antigen.

As used herein, the term “antibody” refers to such assemblies (e.g.,intact antibody molecules, antibody fragments, or variants thereof)which have significant known specific immunoreactive activity to anantigen of interest (e.g. a tumor associated antigen). Antibodies andimmunoglobulins comprise light and heavy chains, with or without aninterchain covalent linkage between them. Basic immunoglobulinstructures in vertebrate systems are relatively well understood.

As will be discussed in more detail below, the generic term “antibody”comprises five distinct classes of antibody that can be distinguishedbiochemically. While all five classes of antibodies are clearly withinthe scope of the current disclosure, the following discussion willgenerally be directed to the IgG class of immunoglobulin molecules. Withregard to IgG, immunoglobulins comprise two identical light chains ofmolecular weight approximately 23,000 Daltons, and two identical heavychains of molecular weight 53,000-70,000. The four chains are joined bydisulfide bonds in a “Y” configuration wherein the light chains bracketthe heavy chains starting at the mouth of the “Y” and continuing throughthe variable region.

Light chains of immunoglobulin are classified as either kappa or lambda(κ, λ). Each heavy chain class may be bound with either a kappa orlambda light chain. In general, the light and heavy chains arecovalently bonded to each other, and the “tail” portions of the twoheavy chains are bonded to each other by covalent disulfide linkages ornon-covalent linkages when the immunoglobulins are generated either byhybridomas, B cells, or genetically engineered host cells. In the heavychain, the amino acid sequences run from an N-terminus at the forkedends of the Y configuration to the C-terminus at the bottom of eachchain. Those skilled in the art will appreciate that heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) withsome subclasses among them (e.g., γ1-γ4). It is the nature of this chainthat determines the “class” of the antibody as IgG, IgM, IgA IgG, orIgE, respectively. The immunoglobulin isotype subclasses (e.g., IgG1,IgG2, IgG3, IgG4, IgA1, etc.) are well characterized and are known toconfer functional specialization. Modified versions of each of theseclasses and isotypes are readily discernable to the skilled artisan inview of the instant disclosure and, accordingly, are within the scope ofthe current disclosure.

Both the light and heavy chains are divided into regions of structuraland functional homology. The term “region” refers to a part or portionof an immunoglobulin or antibody chain and includes constant region orvariable regions, as well as more discrete parts or portions of saidregions. For example, light chain variable regions include“complementarily determining regions” or “CDRs” interspersed among“framework regions” or “FRs”, as defined herein.

The regions of an immunoglobulin heavy or light chain may be defined as“constant” (C) region or “variable” (V) regions, based on the relativelack of sequence variation within the regions of various class membersin the case of a “constant region”, or the significant variation withinthe regions of various class members in the case of a “variableregions”. The terms “constant region” and “variable region” may also beused functionally. In this regard, it will be appreciated that thevariable regions of an immunoglobulin or antibody determine antigenrecognition and specificity. Conversely, the constant regions of animmunoglobulin or antibody confer important effector functions such assecretion, transplacental mobility, Fc receptor binding, complementbinding, and the like. The subunit structures and three dimensionalconfigurations of the constant regions of the various immunoglobulinclasses are well known.

The constant and variable regions of immunoglobulin heavy and lightchains are folded into domains. The term “domain” refers to a globularregion of a heavy or light chain comprising peptide loops (e.g.,comprising 3 to 4 peptide loops) stabilized, for example, by β-pleatedsheet and/or intrachain disulfide bond. Constant region domains on thelight chain of an immunoglobulin are referred to interchangeably as“light chain constant region domains”, “CL regions” or “CL domains”.Constant domains on the heavy chain (e.g. hinge, CH1, CH2 or CH3domains) are referred to interchangeably as “heavy chain constant regiondomains”, “CH” region domains or “CH domains”. Variable domains on thelight chain are referred to interchangeably as “light chain variableregion domains”, “VL region domains or “VL domains” Variable domains onthe heavy chain are referred to interchangeably as “heavy chain variableregion domains”, “VH region domains” or “VH domains”.

By convention the numbering of the variable constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the immunoglobulin or antibody. The N-terminus of eachheavy and light immunoglobulin chain is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively. Accordingly, the domains of a light chain immunoglobulinare arranged in a VL-CL orientation, while the domains of the heavychain are arranged in the VH-CH1-hinge-CH2-CH3 orientation.

Amino acid positions in a heavy chain constant region, including aminoacid positions in the CH1, hinge, CH2, CH3, and CL domains, may benumbered according to the Kabat index numbering system (see Kabat et al,in “Sequences of Proteins of Immunological Interest”, U.S. Dept. Healthand Human Services, 5th edition, 1991). Alternatively, antibody aminoacid positions may be numbered according to the EU index numberingsystem (see Kabat et al, ibid).

As used herein, the term “VH domain” includes the amino terminalvariable domain of an immunoglobulin heavy chain, and the term “VLdomain” includes the amino terminal variable domain of an immunoglobulinlight chain.

As used herein, the term “CH1 domain” includes the first (most aminoterminal) constant region domain of an immunoglobulin heavy chain thatextends, e.g., from about positions 114-223 in the Kabat numberingsystem (EU positions 118-215). The CH1 domain is adjacent to the VHdomain and amino terminal to the hinge region of an immunoglobulin heavychain molecule, and does not form a part of the Fc region of animmunoglobulin heavy chain.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al. J. Immunol.1998, 161:4083).

As used herein, the term “CH2 domain” includes the portion of a heavychain immunoglobulin molecule that extends, e.g., from about positions244-360 in the Kabat numbering system (EU positions 231-340). The CH2domain is unique in that it is not closely paired with another domain.Rather, two N-linked branched carbohydrate chains are interposed betweenthe two CH2 domains of an intact native IgG molecule. In one embodiment,a binding polypeptide of the current disclosure comprises a CH2 domainderived from an IgG1 molecule (e.g. a human IgG1 molecule).

As used herein, the term “CH3 domain” includes the portion of a heavychain immunoglobulin molecule that extends approximately 110 residuesfrom N-terminus of the CH2 domain, e.g., from about positions 361-476 ofthe Kabat numbering system (EU positions 341-445). The CH3 domaintypically forms the C-terminal portion of the antibody. In someimmunoglobulins, however, additional domains may extend from CH3 domainto form the C-terminal portion of the molecule (e.g. the CH4 domain inthe μ chain of IgM and the e chain of IgE). In one embodiment, a bindingpolypeptide of the current disclosure comprises a CH3 domain derivedfrom an IgG1 molecule (e.g. a human IgG1 molecule).

As used herein, the term “CL domain” includes the constant region domainof an immunoglobulin light chain that extends, e.g. from about Kabatposition 107A-216. The CL domain is adjacent to the VL domain. In oneembodiment, a binding polypeptide of the current disclosure comprises aCL domain derived from a kappa light chain (e.g., a human kappa lightchain).

As used herein, the term “Fc region” is defined as the portion of aheavy chain constant region beginning in the hinge region just upstreamof the papain cleavage site (i.e. residue 216 in IgG, taking the firstresidue of heavy chain constant region to be 114) and ending at theC-terminus of the antibody. Accordingly, a complete Fc region comprisesat least a hinge domain, a CH2 domain, and a CH3 domain.

The term “native Fc” as used herein refers to a molecule comprising thesequence of a non-antigen-binding fragment resulting from digestion ofan antibody or produced by other means, whether in monomeric ormultimeric form, and can contain the hinge region. The originalimmunoglobulin source of the native Fc can be of human origin and can beany of the immunoglobulins, such as IgG1 or IgG2. Native Fc moleculesare made up of monomeric polypeptides that can be linked into dimeric ormultimeric forms by covalent (i.e., disulfide bonds) and non-covalentassociation. The number of intermolecular disulfide bonds betweenmonomeric subunits of native Fc molecules ranges from 1 to 4 dependingon class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3,IgA1, and IgGA2). One example of a native Fc is a disulfide-bonded dimerresulting from papain digestion of an IgG. The term “native Fc” as usedherein is generic to the monomeric, dimeric, and multimeric forms.

The term “Fc variant” as used herein refers to a molecule or sequencethat is modified from a native Fc but still comprises a binding site forthe salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fcvariants, and their interaction with the salvage receptor, are known inthe art. Thus, the term “Fc variant” can comprise a molecule or sequencethat is humanized from a non-human native Fc. Furthermore, a native Fccomprises regions that can be removed because they provide structuralfeatures or biological activity that are not required for theantibody-like binding polypeptides. Thus, the term “Fc variant”comprises a molecule or sequence that lacks one or more native Fc sitesor residues, or in which one or more Fc sites or residues has bemodified, that affect or are involved in: (1) disulfide bond formation,(2) incompatibility with a selected host cell, (3) N-terminalheterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC).

The term “Fc domain” as used herein encompasses native Fc and Fcvariants and sequences as defined above. As with Fc variants and nativeFc molecules, the term “Fc domain” includes molecules in monomeric ormultimeric form, whether digested from whole antibody or produced byother means.

As indicated above, the variable regions of an antibody allow it toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain of an antibody combine to form thevariable region (Fv) that defines a three dimensional antigen bindingsite. This quaternary antibody structure forms the antigen binding sitepresent at the end of each arm of the Y. More specifically, the antigenbinding site is defined by three complementary determining regions(CDRs) on each of the heavy and light chain variable regions. As usedherein, the term “antigen binding site” includes a site thatspecifically binds (immunoreacts with) an antigen (e.g., a cell surfaceor soluble antigen). The antigen binding site includes an immunoglobulinheavy chain and light chain variable region and the binding site formedby these variable regions determines the specificity of the antibody. Anantigen binding site is formed by variable regions that vary from oneantibody to another. The altered antibodies of the current disclosurecomprise at least one antigen binding site.

In certain embodiments, binding polypeptides of the current disclosurecomprise at least two antigen binding domains that provide for theassociation of the binding polypeptide with the selected antigen. Theantigen binding domains need not be derived from the same immunoglobulinmolecule. In this regard, the variable region may or be derived from anytype of animal that can be induced to mount a humoral response andgenerate immunoglobulins against the desired antigen. As such, thevariable region of the a binding polypeptide may be, for example, ofmammalian origin e.g., may be human, murine, rat, goat, sheep, non-humanprimate (such as cynomolgus monkeys, macaques, etc.), lupine, or camelid(e.g., from camels, llamas and related species).

In naturally occurring antibodies, the six CDRs present on eachmonomeric antibody are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen binding site as theantibody assumes its three dimensional configuration in an aqueousenvironment. The remainder of the heavy and light variable domains showless inter-molecular variability in amino acid sequence and are termedthe framework regions. The framework regions largely adopt a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope.

Exemplary binding polypeptides featured in the invention includeantibody variants. As used herein, the term “antibody variant” includessynthetic and engineered forms of antibodies which are altered such thatthey are not naturally occurring, e.g., antibodies that comprise atleast two heavy chain portions but not two complete heavy chains (suchas, domain deleted antibodies or minibodies); multispecific forms ofantibodies (e.g., bispecific, trispecific, etc.) altered to bind to twoor more different antigens or to different epitopes on a singleantigen); heavy chain molecules joined to scFv molecules and the like.In addition, the term “antibody variant” includes multivalent forms ofantibodies (e.g., trivalent, tetravalent, etc., antibodies that bind tothree, four or more copies of the same antigen.

As used herein the term “valency” refers to the number of potentialtarget binding sites in a polypeptide. Each target binding sitespecifically binds one target molecule or specific site on a targetmolecule. When a polypeptide comprises more than one target bindingsite, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different ligands or differentantigens, or different epitopes on the same antigen). The subjectbinding polypeptides typically have at least one binding site specificfor a human antigen molecule.

The term “specificity” refers to the ability to specifically bind (e.g.,immunoreact with) a given target antigen (e.g., a human target antigen).A binding polypeptide may be monospecific and contain one or morebinding sites which specifically bind a target or a polypeptide may bemultispecific and contain two or more binding sites which specificallybind the same or different targets. In certain embodiments, a bindingpolypeptide is specific for two different (e.g., non-overlapping)portions of the same target. In certain embodiments, the bindingpolypeptide is specific for more than one target. Exemplary bindingpolypeptides (e.g., antibodies) which comprise antigen binding sitesthat bind to antigens expressed on tumor cells are known in the art andone or more CDRs from such antibodies can be included in an antibody asdescribed herein.

The term “linking moiety” includes moieties which are capable of linkingthe effector moiety to the binding polypeptides disclosed herein. Thelinking moiety may be selected such that it is cleavable (e.g.,enzymatically cleavable or pH-sensitive) or non-cleavable.

As used herein, the term “effector moiety” comprises agents (e.g.proteins, nucleic acids, lipids, carbohydrates, glycopeptides, andfragments thereof) with biological or other functional activity. Forexample, a modified binding polypeptide comprising an effector moietyconjugated to a binding polypeptide has at least one additional functionor property as compared to the unconjugated antibody. For example, theconjugation of a cytotoxic drug (e.g., an effector moiety) to bindingpolypeptide results in the formation of a binding polypeptide with drugcytotoxicity as second function (i.e. in addition to antigen binding).In another example, the conjugation of a second binding polypeptide tothe binding polypeptide may confer additional binding properties. Incertain embodiments, where the effector moiety is a genetically encodedtherapeutic or diagnostic protein or nucleic acid, the effector moietymay be synthesized or expressed by either peptide synthesis orrecombinant DNA methods that are well known in the art. In anotheraspect, where the effector moiety is a non-genetically encoded peptide,or a drug moiety, the effector moiety may be synthesized artificially orpurified from a natural source. As used herein, the term “drug moiety”includes anti-inflammatory, anticancer, anti-infective (e.g.,anti-fungal, antibacterial, anti-parasitic, anti-viral, etc.), andanesthetic therapeutic agents. In a further embodiment, the drug moietyis an anticancer or cytotoxic agent. Compatible drug moieties may alsocomprise prodrugs. Exemplary effector moieties are set forth in Table 1herein.

In certain embodiments, an “effector moiety” comprises a “targetingmoiety.” As used herein, the term “targeting moiety” refers to aneffector moiety that binds to a target molecule. Targeting moieties cancomprise, without limitation, proteins, nucleic acids, lipids,carbohydrates (e.g., glycans), and combinations thereof (e.g.,glycoproteins, glycopeptides, and glycolipids).

As used herein, the term “prodrug” refers to a precursor or derivativeform of a pharmaceutically active agent that is less active, reactive orprone to side effects as compared to the parent drug and is capable ofbeing enzymatically activated or otherwise converted into a more activeform in vivo. Prodrugs compatible with the compositions of the currentdisclosure include, but are not limited to, phosphate-containingprodrugs, amino acid-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs that can be converted to the more activecytotoxic free drug. One skilled in the art may make chemicalmodifications to the desired drug moiety or its prodrug in order to makereactions of that compound more convenient for purposes of preparingmodified binding polypeptides of the current disclosure. The drugmoieties also include derivatives, pharmaceutically acceptable salts,esters, amides, and ethers of the drug moieties described herein.Derivatives include modifications to drugs identified herein which mayimprove or not significantly reduce a particular drug's desiredtherapeutic activity.

As used herein, the term “anticancer agent” includes agents which aredetrimental to the growth and/or proliferation of neoplastic or tumorcells and may act to reduce, inhibit or destroy malignancy. Examples ofsuch agents include, but are not limited to, cytostatic agents,alkylating agents, antibiotics, cytotoxic nucleosides, tubulin bindingagents, hormones, hormone antagonists, cytotoxic agents, and the like.Cytotoxic agents include tomaymycin derivatives, maytansine derivatives,cryptophycine derivatives, anthracycline derivatives, bisphosphonatederivatives, leptomycin derivatives, streptonigrin derivatives,auristatine derivatives, and duocarmycin derivatives. Any agent thatacts to retard or slow the growth of immunoreactive cells or malignantcells is within the scope of the current disclosure.

The term “antigen” or “target antigen” as used herein refers to amolecule or a portion of a molecule that is capable of being bound bythe binding site of a binding polypeptide. A target antigen may have oneor more epitopes.

The term “sialic acid derivative-conjugated binding polypeptide” as usedherein refers to the polypeptide formed by reacting a CMP-sialic acidderivative with a glycan of a binding peptide. For example, a sialicacid derivative-conjugated binding polypeptide includes, but is notlimited to, polypeptides of FIGS. 3A-E represented by circles incombination with the reactive moieties to which they are bonded.

The term “trivalent glycopeptide” as used herein refers to a targetingor effector moiety comprising three glycopeptides.

The term “trivalent aminooxy,” as used herein, refers to an aninooxymoiety further comprising three carbohydrates or glycopeptides. Thetrivalent aminooxy may contain additional functional groups, e.g., alinker.

As used herein, “click chemistry” refers to pairs of terminal reactivemoieties that rapidly and selectively react (“click”) with each other toform a targeting or effector moiety conjugated binding polypeptide.Click chemistry is discussed further herein.

As used herein, the term “metal catalyst” refers to catalysts thatcomprise a transition metal including, but not limited to, ruthenium,nickel, palladium, platinum, and iron and one or more ligands including,but not limited to, bipyridine derivatives or terpyridine derivatives. Ametal catalyst may also be formed in situ. For example, a copper(II)compound may be added to the reaction mixture in the presence of areducing agent including, but not limited to, copper sulfate (CuSO₄) asthe copper(II) compound and sodium ascorbate as the reducing agent.

As used herein, the term “reactive moiety” refers to a moiety comprisinga portion or an entire functional group are specific groups of one ormore atoms and one or more bonds that are responsible for characteristicchemical reactions. In example embodiments, a reactive moiety includes,but is not limited to, an aldehyde moiety, an alkyne, an aminooxymoiety, an azide, a hydrazine, a keto moiety, and a thiol. In someembodiments, the reactive moiety is a terminal reactive moiety. In thereacting step, a first reactive moiety reacts with a second reactivemoiety to form an effector moiety conjugated binding polypeptide.

An “aldehyde” moiety, as used herein, refers to a formyl functionalgroup and is represented by the following structural formula:

For example, a CMP-sialic acid-derivative comprising a terminal aldehydemoiety includes, but is not limited to, the following structuralformulas:

An “alkyne” moiety, as used herein, refers to a carbon-carbon triplebond.

An “aminooxy” moiety, as used herein, refers to a nitrogen-oxygen singlebond and is represented by the following structural formula:

An “azide” moiety, as used herein, refers to an RN₃ moiety and may berepresented by the following structural formula:

A “hydrazine” moiety, as used herein, refers to at least onenitrogen-nitrogen single bond and is represented by the followingstructural formula:

For example, a hydrazine may have a structural formula of:

As used herein, an “imine” moiety refers to a carbon-nitrogen doublebond and is represented by the following structural formula:

In some embodiments, a targeting or effector moiety conjugated bindingpolypeptide comprises an imine. For example, a type of imine includes,but is not limited to, an aldimine, a hydroxylamine, a hydrazone, aketamine, or an oxime.

A “hydrazone” moiety, as used herein, refers to a type of imine and isrepresented by the following structural formula:

In some embodiments, the hydrazone may be a terminal hydrazone. In someembodiments, a hydrazone linkage comprises a hydrazone moiety along withadditional functional groups, e.g., a linker or a portion of a linkingmoiety.

A “keto” or “ketone” moiety, as used herein, comprises a carbonylfunctional group and is represented by the following structural formula:

A “maleimide” moiety, as used herein, comprises an unsaturated imide andis represented by the following structural formula:

An “oxime” moiety is a type of imine and is represented by the followingstructural formula:

The term “thioether” is represented by the following structural formula:

A “thiol” refers to a moiety comprising a —SH functional group, which isalso referred to as a sulfhydryl group. In some embodiments, a thiolcontains a carbon-bonded sulfhydryl group.

The term “terminal” when referring to a reactive moiety, as used herein,describes a group bonded to a terminus of a straight or branched-chainmoiety. In some embodiments, the terminal reactive moiety is asubstituent of a functional group.

The term “oxidizing agent” refers to a compound or a reagent thataccepts or gains electrons from another compound or reagent therebyundergoing a reduction while oxidizing the other compound or reagent.For example, oxidizing agents include, but are not limited to, sodiumperiodate, periodate oxidase, galactose oxidase, hydrogen peroxide, andcopper compounds (e.g., copper(II) sulfate).

The term “ambient temperature,” as used herein, is equivalent to theterm “room temperature” and denotes the range of temperatures between20° C. and 26° C. (equivalent to 68° F. and 79° F.), with an averagetemperature of approximately of 23° C. (73° F.).

The term “effector moiety conjugated binding polypeptide,” as usedherein, refers to a structure comprising one or more binding proteinslinked or bonded to an effector moiety. There may be a number ofchemical moieties and functional groups that comprise the linkagebetween the binding protein(s) and the effector moieties(s) including,but not limited to, any glycan or modified glycan (e.g. one or moresialic acid derivatives or CMP-sialic acid derivatives).

II. Sialic Acid Derivatives

In one aspect, the current disclosure provides for a method of makingsialic acids or sialic acid derivatives from sugars or sugarderivatives. The sugar or sugar derivative used may be but is notlimited to N-acetylmannosamine or its derivatives such as N-acetylmannosamine (ManNAc), N-levulinoyl mannosamine (ManLev),N-azidoacetylmannosamine (ManHAz), azidomannosamine, and N-thioacetylmannosamine (ManHS).

In example embodiments, the sugar or sugar derivative has the followingstructural formula:

wherein R₁ is a reactive moiety including, but not limited to,NH(C═O)CH₃, NH(C═O)CH₂CH₂(C═O)CH₃, NH(C═O)CH₂OH, NH(C═O)CH₂N₃,NH(C═O)SH, OH or N₃.

In some embodiments, the CMP-sialic acid derivative has the followingstructural formula:

wherein R1 is a reactive moiety including, but not limited to, thegroups listed above.

III. Binding Polypeptides

In one aspect, the current disclosure provides binding polypeptides(e.g., antibodies, antibody fragments, antibody variants, and fusionproteins) comprising a glycosylated domain, e.g., a glycosylatedconstant domain. The binding polypeptides disclosed herein encompass anybinding polypeptide that comprises a domain having an N-linkedglycosylation site. In certain embodiments, the binding polypeptide isan antibody, or fragment or derivative thereof. Any antibody from anysource or species can be employed in the binding polypeptides disclosedherein. Suitable antibodies include without limitation, humanantibodies, humanized antibodies or chimeric antibodies.

In certain embodiments, the glycosylated domain is an Fc domain. Incertain embodiments, the glycosylation domain is a native glycosylationdomain at N297.

In other embodiments, the glycosylation domain is an engineeredglycosylation domain. Exemplary engineered glycosylation domains in Fcdomain comprise an asparagine residue at amino acid position 298,according to EU numbering; and a serine or threonine residue at aminoacid position 300, according to EU numbering.

Fc domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA andIgE) and species can be used in the binding polypeptides disclosedherein. Chimeric Fc domains comprising portions of Fc domains fromdifferent species or Ig classes can also be employed. In certainembodiments, the Fc domain is a human IgG1 Fc domain. In the case of ahuman IgG1 Fc domain, mutation of the wild type amino acid at Kabatposition 298 to an asparagine and Kabat position 300 to a serine orthreonine results in the formation of an N-linked glycosylationconsensus site (i.e, the N-X-T/S sequon, where X is any amino acidexcept proline). However, in the case of Fc domains of other speciesand/or Ig classes or isotypes, the skill artisan will appreciate that itmay be necessary to mutate Kabat position 299 of the Fc domain if aproline residue is present to recreate an N-X-T/S sequon.

In other embodiments, the current disclosure provides bindingpolypeptides (e.g., antibodies, antibody fragments, antibody variants,and fusion proteins) comprising at least one CH1 domain having anN-linked glycosylation site. Such exemplary binding polypeptides includemay comprise, for example, and engineered glycosylation site at position114, according to Kabat numbering.

CH1 domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA andIgE) and species can be used in the binding polypeptides disclosedherein. Chimeric CHI domains comprising portions of CHI domains fromdifferent species or Ig classes can also be employed. In certainembodiments, the CH1 domain is a human IgG1 CH1 domain. In the case of ahuman IgG1 domain, mutation of the wild type amino acid at position 114to an asparagine results in the formation of an N-linked glycosylationconsensus site (i.e, the N-X-T/S sequon, where X is any amino acidexcept proline). However, in the case of other CH1 domains of otherspecies and/or Ig classes or isotypes, the skilled artisan willappreciate that it may be necessary to mutate positions 115 and/or 116of the CH1 domain to create an N-X-T/S sequon.

In certain embodiments, the binding polypeptide of the currentdisclosure may comprise an antigen binding fragment of an antibody. Theterm “antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody which binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). Antigen binding fragmentscan be produced by recombinant or biochemical methods that are wellknown in the art. Exemplary antigen-binding fragments include Fv, Fab,Fab′, and (Fab′)2. In some embodiments, the antigen-binding fragment ofthe current disclosure is an altered antigen-binding fragment comprisingat least one engineered glycosylation site. In one exemplary embodiment,an altered antigen binding fragment of the current disclosure comprisesan altered VH domain described supra. In another exemplary embodiment,an altered antigen binding fragment of the current disclosure comprisesan altered CH1 domain described supra.

In exemplary embodiments, the binding polypeptide comprises a singlechain variable region sequence (ScFv). Single chain variable regionsequences comprise a single polypeptide having one or more antigenbinding sites, e.g., a VL domain linked by a flexible linker to a VHdomain ScFv molecules can be constructed in a VH-linker-VL orientationor VL-linker-VH orientation. The flexible hinge that links the VL and VHdomains that make up the antigen binding site typically has from about10 to about 50 amino acid residues. Connecting peptides are known in theart. Binding polypeptides may comprise at least one scFv and/or at leastone constant region. In one embodiment, a binding polypeptide of thecurrent disclosure may comprise at least one scFv linked or fused to anantibody or fragment comprising a CH1 domain (e.g. a CH1 domaincomprising an asparagine residue at Kabat position 114) and/or a CH2domain (e.g. a CH2 domain comprising an asparagine residue at EUposition 298, and a serine or threonine residue at EU position 300).

In certain exemplary embodiments, a binding polypeptide of the currentdisclosure is a multivalent (e.g., tetravalent) antibody which isproduced by fusing a DNA sequence encoding an antibody with a ScFvmolecule (e.g., an altered ScFv molecule). For example, in oneembodiment, these sequences are combined such that the ScFv molecule(e.g., an altered ScFv molecule) is linked at its N-terminus orC-terminus to an Fc fragment of an antibody via a flexible linker (e.g.,a gly/ser linker). In another embodiment a tetravalent antibody of thecurrent disclosure can be made by fusing an ScFv molecule to aconnecting peptide, which is fused to a CH1 domain (e.g. a CH1 domaincomprising an asparagine residue at Kabat position 114) to construct anScFv-Fab tetravalent molecule.

In another embodiment, a binding polypeptide of the current disclosureis an altered minibody. Altered minibodies of the current disclosure aredimeric molecules made up of two polypeptide chains each comprising anScFv molecule (e.g., an altered ScFv molecule comprising an altered VHdomain described supra) which is fused to a CH3 domain or portionthereof via a connecting peptide. Minibodies can be made by constructingan ScFv component and connecting peptide-CH3 components using methodsdescribed in the art (see, e.g., U.S. Pat. No. 5,837,821 or WO94/09817A1). In another embodiment, a tetravalent minibody can beconstructed. Tetravalent minibodies can be constructed in the samemanner as minibodies, except that two ScFv molecules are linked using aflexible linker. The linked scFv-scFv construct is then joined to a CH3domain.

In another embodiment, a binding polypeptide of the current disclosurecomprises a diabody. Diabodies are dimeric, tetravalent molecules eachhaving a polypeptide similar to scFv molecules, but usually having ashort (less than 10, e.g., 1-5) amino acid residue linker connectingboth variable domains, such that the VL and VH domains on the samepolypeptide chain cannot interact. Instead, the VL and VH domain of onepolypeptide chain interact with the VH and VL domain (respectively) on asecond polypeptide chain (see, for example, WO 02/02781). Diabodies ofthe current disclosure comprise an scFv molecule fused to a CH3 domain.

In other embodiments, the binding polypeptides include multispecific ormultivalent antibodies comprising one or more variable domain in serieson the same polypeptide chain, e.g., tandem variable domain (TVD)polypeptides. Exemplary TVD polypeptides include the “double head” or“Dual-Fv” configuration described in U.S. Pat. No. 5,989,830. In theDual-Fv configuration, the variable domains of two different antibodiesare expressed in a tandem orientation on two separate chains (one heavychain and one light chain), wherein one polypeptide chain has two VHdomains in series separated by a peptide linker (VH1-linker-VH2) and theother polypeptide chain consists of complementary VL domains connectedin series by a peptide linker (VL1-linker-VL2). In the cross-over doublehead configuration, the variable domains of two different antibodies areexpressed in a tandem orientation on two separate polypeptide chains(one heavy chain and one light chain), wherein one polypeptide chain hastwo VH domains in series separated by a peptide linker (VH1-linker-VH2)and the other polypeptide chain consists of complementary VL domainsconnected in series by a peptide linker in the opposite orientation(VL2-linker-VL1). Additional antibody variants based on the “Dual-Fv”format include the Dual-Variable-Domain IgG (DVD-IgG) bispecificantibody (see U.S. Pat. No. 7,612,181 and the TBTI format (see US2010/0226923 A1). The addition of constant domains to respective chainsof the Dual-Fv (CH1-Fc to the heavy chain and kappa or lambda constantdomain to the light chain) leads to functional bispecific antibodieswithout any need for additional modifications (i.e., obvious addition ofconstant domains to enhance stability).

In another exemplary embodiment, the binding polypeptide comprises across-over dual variable domain IgG (CODV-IgG) bispecific antibody basedon a “double head” configuration (see US20120251541 A1, which isincorporated by reference herein in its entirety). CODV-IgG antibodyvariants have one polypeptide chain with VL domains connected in seriesto a CL domain (VL1-L1-VL2-L2-CL) and a second polypeptide chain withcomplementary VH domains connected in series in the opposite orientationto a CH1 domain (VH2-L3-VH1-L4-CH1), where the polypeptide chains form across-over light chain-heavy chain pair. In certain embodiment, thesecond polypeptide may be further connected to an Fc domain(VH2-L3-VH1-L4-CH1-Fc). In certain embodiments, linker L3 is at leasttwice the length of linker L1 and/or linker L4 is at least twice thelength of linker L2. For example, L1 and L2 may be 1-3 amino acidresidues in length, L3 may be 2 to 6 amino acid residues in length, andL4 may be 4 to 7 amino acid residues in length. Examples of suitablelinkers include a single glycine (Gly) residue; a diglycine peptide(Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycineresidues (Gly-Gly-Gly-Gly (SEQ ID NO: 17)); a peptide with five glycineresidues (Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 18)); a peptide with sixglycine residues (Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 19)); a peptidewith seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO:20)); a peptide with eight glycine residues(Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly (SEQ ID NO: 21)). Other combinations ofamino acid residues may be used such as the peptide Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 22) and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 39).

In certain embodiments, the binding polypeptide comprises animmunoadhesin molecule comprising a non-antibody binding region (e.g., areceptor, ligand, or cell-adhesion molecule) fused to an antibodyconstant region (see e.g., Ashkenazi et al., Methods, 1995 8(2),104-115, which is incorporated by reference herein in its entirety)

In certain embodiments, the binding polypeptide comprisesimmunoglobulin-like domains. Suitable immunoglobulin-like domainsinclude, without limitation, fibronectin domains (see, for example,Koide et al. (2007), Methods Mol. Biol. 352: 95-109, which isincorporated by reference herein in its entirety), DARPin (see, forexample, Stumpp et al. (2008) Drug Discov. Today 13 (15-16): 695-701,which is incorporated by reference herein in its entirety), Z domains ofprotein A (see, Nygren et al. (2008) FEBS J. 275 (11): 2668-76, which isincorporated by reference herein in its entirety), Lipocalins (see, forexample, Skerra et al. (2008) FEBS J. 275 (11): 2677-83, which isincorporated by reference herein in its entirety), Affilins (see, forexample, Ebersbach et al. (2007) J. Mol. Biol. 372 (1): 172-85, which isincorporated by reference herein in its entirety), Affitins (see, forexample, Krehenbrink et al. (2008). J. Mol. Biol. 383 (5): 1058-68,which is incorporated by reference herein in its entirety), Avimers(see, for example, Silverman et al. (2005) Nat. Biotechnol. 23 (12):1556-61, which is incorporated by reference herein in its entirety),Fynomers, (see, for example, Grabulovski et al. (2007) J Biol Chem 282(5): 3196-3204, which is incorporated by reference herein in itsentirety), and Kunitz domain peptides (see, for example, Nixon et al.(2006) Curr Opin Drug Discov Devel 9 (2): 261-8, which is incorporatedby reference herein in its entirety).

IV. N-Linked Glycans

In certain embodiments, the binding polypeptides employs N-linkedglycans which are “N-linked” via an asparagine residue to aglycosylation site in the polypeptide backbone of the bindingpolypeptide. The glycosylation site may be a native or engineeredglycosylation site. Additionally or alternatively, the glycan may be anative glycan or an engineered glycan containing non-native linkages.

In certain exemplary embodiments, the binding polypeptide includes thenative glycosylation site of an antibody Fc domain. This nativeglycosylation site comprises a wild-type asparagine residue at position297 of the Fc domain (N297), according to EU numbering. The nativeN-linked glycan that resides at this position is generally linked thougha (3-glycosylamide linkage to the nitrogen group of the N297 side chain.However, other suitable art recognized linkages can also be employed. Inother exemplary embodiments, the binding polypeptides comprise one ormore engineered glycosylation sites. Such engineered glycosylation sitescomprise the substitution of one or more wild-type amino acids in thepolypeptide backbone of the binding polypeptide with an asparagineresidue that is capable of being N-glycosylated by the glycosylationenzymes of a cell. Exemplary engineered glycosylation sites include theintroduction of asparagine mutation at amino acid position 298 of the Fcdomain (298N) or amino acid position 114 of a CH1 domain (114N).

Any type of naturally occurring or synthetic (i.e., non-natural)N-linkedglycan can be linked to a glycosylation site of a binding polypeptidefeatured in the invention. In certain embodiments, the glycan comprisesa saccharide (e.g., a saccharide residue located at terminus of anoligosaccharide) that can be oxidized (e.g., by periodate treatment orgalactose oxidase) to produce a group suitable for conjugation to aneffector moiety (e.g., a reactive aldehyde group). Suitable oxidizablesaccharides included, without limitation, galactose and sialic acid(e.g., N-Acetylneuraminic acid). In other embodiments, the glycancomprises a sialic acid or sialic acid derivative that does not requirefurther oxidation to produce a group suitable for conjugation to aneffector moiety (e.g., a reactive moiety including, but not limited to,an aldehyde moiety, an alkyne, an aminooxy moiety, an azide, ahydrazine, a keto moiety, and a thiol). In specific embodiments, theglycan comprises a sialic acid derivative. In one embodiment, the glycancomprising a sialic acid derivative is formed by a reaction between abinding polypeptide comprising a glycan and a CMP-sialic acidderivative. In one embodiment, the sialic acid derivative or CMP-sialicacid derivative may comprise a terminal azide moiety. In a furtherembodiment, the CMP-sialic acid derivative may be a CMP-sialic acid C5azide. In another embodiment, the sialic acid derivative may comprise aC5 azide. In certain embodiments, the CMP-sialic acid derivative has thefollowing structural formula:

wherein R₁ is a reactive moiety including, but not limited to,NH(C═O)CH3, NH(C═O)CH₂CH₂(C═O)CH₃, NH(C═O)CH₂OH, NH(C═O)CH₂N₃,NH(C═O)SH, OH or N₃.

In certain embodiments, the glycan is a biantennary glycan. In certainembodiments, the glycan is a naturally occurring mammalian glycoform.

Glycosylation can be achieved through any means known in the art. Incertain embodiments, the glycosylation is achieved by expression of thebinding polypeptides in cells capable of N-linked glycosylation. Anynatural or engineered cell (e.g., prokaryotic or eukaryotic) can beemployed. In general, mammalian cells are employed to effectglycosylation. The N-glycans that are produced in mammalian cells arecommonly referred to as complex, high manose, hybrid-type N-glycans (seee.g., Drickamer K, Taylor M E (2006). Introduction to Glycobiology, 2nded., which is incorporated herein by reference in its entirety). Thesecomplex N-glycans have a structure that typically has two to six outerbranches with a sialyllactosamine sequence linked to an inner corestructure Man₃GlcNAc₂. A complex N-glycan has at least one branch, e.g.,at least two, of alternating GlcNAc and galactose (Gal) residues thatterminate in oligosaccharides such as, for example: NeuNAc-; NeuAc α2,6GalNAc α1-; NeuAc α2,3 Gal β1,3 GalNAc α1-; and NeuAc α2,3/6 Gal β1,4GlcNAc β1. In addition, sulfate esters can occur on galactose, GalNAc,and GlcNAc residues. NeuAc can be O-acetylated or replaced by NeuG1(N-glycolylneuraminic acid). Complex N-glycans may also have intrachainsubstitutions of bisecting GlcNAc and core fucose (Fuc).

Additionally or alternatively, glycosylation can be achieved or modifiedthrough enzymatic means, in vitro. For example, one or moreglycosyltransferases may be employed to add specific saccharide residuesto the native or engineered N-glycan of a binding polypeptide, and oneor more glycosidases may be employed to remove unwanted saccharides fromthe N-linked glycan. Such enzymatic means are well known in the art(see. e.g., WO2007/005786, which is incorporated herein by reference inits entirety).

V. Immunological Effector Functions and Fc Modifications

In certain embodiments, binding polypeptides may include an antibodyconstant region (e.g. an IgG constant region e.g., a human IgG constantregion, e.g., a human IgG1 or IgG4 constant region) which mediates oneor more effector functions. For example, binding of the C1-complex to anantibody constant region may activate the complement system. Activationof the complement system is important in the opsonisation and lysis ofcell pathogens. The activation of the complement system also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region (Fc receptor binding sites on the antibody Fc regionbind to Fc receptors (FcRs) on a cell). There are a number of Fcreceptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production. In some embodiments,the binding polypeptides (e.g., antibodies or antigen binding fragmentsthereof) featured in the invention bind to an Fc-gamma receptor. Inalternative embodiments, binding polypeptides may include a constantregion which is devoid of one or more effector functions (e.g., ADCCactivity) and/or is unable to bind Fcγ receptor.

Certain embodiments include antibodies in which at least one amino acidin one or more of the constant region domains has been deleted orotherwise altered so as to provide desired biochemical characteristicssuch as reduced or enhanced effector functions, the ability tonon-covalently dimerize, increased ability to localize at the site of atumor, reduced serum half-life, or increased serum half-life whencompared with a whole, unaltered antibody of approximately the sameimmunogenicity. For example, certain antibodies for use in thediagnostic and treatment methods described herein are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the CH2 domain will be deleted.

In certain other embodiments, binding polypeptides comprise constantregions derived from different antibody isotypes (e.g., constant regionsfrom two or more of a human IgG1, IgG2, IgG3, or IgG4). In otherembodiments, binding polypeptides comprises a chimeric hinge (i.e., ahinge comprising hinge portions derived from hinge domains of differentantibody isotypes, e.g., an upper hinge domain from an IgG4 molecule andan IgG1 middle hinge domain). In one embodiment, binding polypeptidescomprise an Fc region or portion thereof from a human IgG4 molecule anda Ser228Pro mutation (EU numbering) in the core hinge region of themolecule.

In certain embodiments, the Fc portion may be mutated to increase ordecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or flexibility. Theresulting physiological profile, bioavailability and other biochemicaleffects of the modifications, such as tumor localization,biodistribution and serum half-life, may easily be measured andquantified using well know immunological techniques without undueexperimentation.

In certain embodiments, an Fc domain employed in an antibody featured inthe invention is an Fc variant. As used herein, the term “Fc variant”refers to an Fc domain having at least one amino acid substitutionrelative to the wild-type Fc domain from which said Fc domain isderived. For example, wherein the Fc domain is derived from a human IgG1antibody, the Fc variant of said human IgG1 Fc domain comprises at leastone amino acid substitution relative to said Fc domain.

The amino acid substitution(s) of an Fc variant may be located at anyposition (i.e., any EU convention amino acid position) within the Fcdomain. In one embodiment, the Fc variant comprises a substitution at anamino acid position located in a hinge domain or portion thereof. Inanother embodiment, the Fc variant comprises a substitution at an aminoacid position located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

The binding polypeptides may employ any art-recognized Fc variant whichis known to impart an improvement (e.g., reduction or enhancement) ineffector function and/or FcR binding. Said Fc variants may include, forexample, any one of the amino acid substitutions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2 or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250;5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375;6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784,each of which is incorporated in its entirety by reference herein. Inone exemplary embodiment, a binding polypeptide may comprise an Fcvariant comprising an amino acid substitution at EU position 268 (e.g.,H268D or H268E). In another exemplary embodiment, a binding polypeptidemay include an amino acid substitution at EU position 239 (e.g., S239Dor S239E) and/or EU position 332 (e.g., I332D or I332Q).

In certain embodiments, a binding polypeptide may include an Fc variantcomprising an amino acid substitution which alters theantigen-independent effector functions of the antibody, in particularthe circulating half-life of the binding polypeptide. Such bindingpolypeptides exhibit either increased or decreased binding to FcRn whencompared to binding polypeptides lacking these substitutions, therefore,have an increased or decreased half-life in serum, respectively. Fcvariants with improved affinity for FcRn are anticipated to have longerserum half-lives, and such molecules have useful applications in methodsof treating mammals where long half-life of the administered antibody isdesired, e.g., to treat a chronic disease or disorder. In contrast, Fcvariants with decreased FcRn binding affinity are expected to haveshorter half-lives, and such molecules are also useful, for example, foradministration to a mammal where a shortened circulation time may beadvantageous, e.g. for in vivo diagnostic imaging or in situations wherethe starting antibody has toxic side effects when present in thecirculation for prolonged periods. Fc variants with decreased FcRnbinding affinity are also less likely to cross the placenta and, thus,are also useful in the treatment of diseases or disorders in pregnantwomen. In addition, other applications in which reduced FcRn bindingaffinity may be desired include applications localized to the brain,kidney, and/or liver. In one exemplary embodiment, the altered bindingpolypeptides (e.g., antibodies or antigen binding fragments thereof)exhibit reduced transport across the epithelium of kidney glomeruli fromthe vasculature. In another embodiment, the altered binding polypeptides(e.g., antibodies or antigen binding fragments thereof) exhibit reducedtransport across the blood brain barrier (BBB) from the brain into thevascular space. In one embodiment, an antibody with altered FcRn bindingcomprises an Fc domain having one or more amino acid substitutionswithin the “FcRn binding loop” of an Fc domain. The FcRn binding loop iscomprised of amino acid residues 280-299 (according to EU numbering).Exemplary amino acid substitutions which alter FcRn binding activity aredisclosed in International PCT Publication No. WO05/047327 which isincorporated in its entirety by reference herein. In certain exemplaryembodiments, the binding polypeptides (e.g., antibodies or antigenbinding fragments thereof) include an Fc domain having one or more ofthe following substitutions: V284E, H285E, N286D, K290E and S304D (EUnumbering). In yet other exemplary embodiments, the binding moleculesinclude a human Fc domain with the double mutation H433K/N434F (see,e.g., U.S. Pat. No. 8,163,881).

In other embodiments, binding polypeptides, for use in the diagnosticand treatment methods described herein have a constant region, e.g., anIgG1 or IgG4 heavy chain constant region, which is altered to reduce oreliminate glycosylation. For example, binding polypeptides (e.g.,antibodies or antigen binding fragments thereof) may also include an Fcvariant comprising an amino acid substitution which alters theglycosylation of the antibody Fc. For example, said Fc variant may havereduced glycosylation (e.g., N- or O-linked glycosylation). In exemplaryembodiments, the Fc variant comprises reduced glycosylation of theN-linked glycan normally found at amino acid position 297 (EUnumbering). In another embodiment, the antibody has an amino acidsubstitution near or within a glycosylation motif, for example, anN-linked glycosylation motif that contains the amino acid sequence NXTor NXS. In a particular embodiment, the antibody comprises an Fc variantwith an amino acid substitution at amino acid position 228 or 299 (EUnumbering). In more particular embodiments, the antibody comprises anIgG1 or IgG4 constant region comprising an S228P and a T299A mutation(EU numbering).

Exemplary amino acid substitutions which confer reduced or alteredglycosylation are disclosed in International PCT Publication No.WO05/018572, which is incorporated in its entirety by reference herein.In some embodiments, the binding polypeptides are modified to eliminateglycosylation. Such binding polypeptides may be referred to as “agly”binding polypeptides (e.g. “agly” antibodies). While not being bound bytheory, it is believed that “agly” binding polypeptides may have animproved safety and stability profile in vivo. Agly binding polypeptidescan be of any isotype or subclass thereof, e.g., IgG1, IgG2, IgG3, orIgG4. In certain embodiments, agly binding polypeptides comprise anaglycosylated Fc region of an IgG4 antibody which is devoid ofFc-effector function, thereby eliminating the potential for Fc mediatedtoxicity to the normal vital organs that express IL-6. In yet otherembodiments, binding polypeptides include an altered glycan. Forexample, the antibody may have a reduced number of fucose residues on anN-glycan at Asn297 of the Fc region, i.e., is afucosylated.Afucosylation increases FcγRII binding on the NK cells and potentlyincreases ADCC. It has been shown that a diabody comprising an anti-IL-6scFv and an anti-CD3 scFv induces killing of IL-6 expressing cells byADCC. Accordingly, in one embodiment, an afucosylated anti-IL-6 antibodyis used to target and kill IL-6-expressing cells. In another embodiment,the binding polypeptide may have an altered number of sialic acidresidues on the N-glycan at Asn297 of the Fc region. Numerousart-recognized methods are available for making “agly” antibodies orantibodies with altered glycans. For example, genetically engineeredhost cells (e.g., modified yeast, e.g., Picchia, or CHO cells) withmodified glycosylation pathways (e.g., glycosyl-transferase deletions)can be used to produce such antibodies.

VI. Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure comprise effector moieties (e.g., targeting moieties). Ingeneral these effector moieties are conjugated (either directly orthrough a linker moiety) to an N-linked glycan on the bindingpolypeptide, (e.g., an N-linked glycan linked to N298 (EU numbering) ofthe CH2 domain and/or N114 (Kabat numbering) of a CH1 domain). Incertain embodiments, the binding polypeptide is full length antibodycomprising two CH1 domains with a glycan at Kabat position 114, whereinboth of the glycans are conjugated to one or more effector moieties.

Any effector moiety can be added to the binding polypeptides disclosedherein. The effector moieties typically add a non-natural function to analtered antibody or fragments thereof without significantly altering theintrinsic activity of the binding polypeptide. The effector moiety maybe, for example but not limited to, targeting moiety (e.g., aglycopeptide or neoglycan). A modified binding polypeptide (e.g., anantibody) of the current disclosure may comprise one or more effectormoieties, which may be the same of different.

In one embodiment, the effector moiety can be of Formula (I):H₂N-Q-CON—X   Formula (I),wherein:

A) Q is NH or O; and

B) CON is a connector moiety; and

C) X is an effector moiety (e.g., a targeting moiety as defined herein).

The connector moiety connects the therapeutic agent to H₂N-Q-. Theconnector moiety can include at least one of any suitable componentsknown to those skilled in the art, including, for example, an alkylenylcomponent, a polyethylene glycol component, a poly(glycine) component, apoly(oxazoline) component, a carbonyl component, a component derivedfrom cysteinamide, a component derived from valine coupled withcitruline, and a component derived from 4-aminobenzyl carbamate, or anycombination thereof.

In some embodiments, the connector moiety (CON) may comprise portions ofthe molecules formed in the reacting step whereby an effector moietyconjugated binding polypeptide is formed. For example, the connectormoiety may comprise one or more of the following structural formulas:

In another embodiment, the effector moiety of Formula (I) can be ofFormula (Ia):H₂N-Q-CH₂—C(O)—Z—X   Formula (Ia),wherein:

A) Q is NH or O; and

B) Z is -Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f),

-   -   wherein        -   i. Cys is a component derived cysteinamide;        -   ii. MC is a component derived from maleimide;        -   iii. VC is a component derived from valine coupled with            citruline;        -   iv. PABC is a component derived from 4-aminobenzyl            carbamate;        -   v. X is an effector moiety (e.g., a targeting moiety as            defined herein);        -   vi. a is 0 or 1;        -   vii. b is 0 or 1;        -   viii. c is 0 or 1; and        -   ix. f is 0 or 1

The “component derived from cysteinamide” is the point of attachment toH₂N-Q-CH₂—C(O)—. In one embodiment, the “component derived fromcysteinamide” can refer to one or more portions of the effector moietyhaving the structure:

In one embodiment, the “Cys” component of an effector moiety may includeone such portion. For example, the following structure shows an effectormoiety with one such portion (wherein the “Cys” component is indicatedwith the dotted line box):

In another embodiment, the “Cys” component of an effector moiety mayinclude two or more such portions. For example, the following moietycontains two such portions:

As can be seen from the structure, each “Cys” component bears an-(MC)_(a)(VC)_(b)-(PABC)_(c)—(C₁₆H₃₂O₈C₂H₄)_(f)—X group.

In one embodiment, the phrase “component derived from maleimide” canrefer to any portion of the effector moiety having the structure:

wherein d is an integer from 2 to 5. The number of MC componentsincluded in any Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—Xgroup in the effector moiety is indicated by subscript “a,” and can be 0or 1 In one embodiment, a is 1. In another embodiment, b is 0.

In one embodiment, the “Cys” component can be connected to the “MC”component via the sulfur atom in the “Cys” component, as indicated withthe dotted line box in the structure below:

In one embodiment, the phrase “component derived from valine coupledwith citruline” can refer to any portion of the effector moiety with thefollowing structure:

The number of VC components included in anyCys-(MC)_(a)(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H4)_(f)—X group in theeffector moiety is indicated by subscript “b,” and can be 0 or 1. In oneembodiment, b is 1. In another embodiment, b is 0.

In one embodiment, the phrase “component derived from 4-aminobenzylcarbamate” can refer to any portion of the effector moiety with thefollowing structure:

The number of PABC components included in anyCys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—X group in theeffector moiety is indicated by subscript “c,” and can be 0 or 1. In oneembodiment, c is 1. In another embodiment, c is 0.

In one embodiment, “C₁₆H₃₂O₈C₂H₄” refers to the following structure:

The number of C₁₆H₃₂O₈ units included in anyCys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—X group in theeffector moiety is indicated by subscript “f,” In one embodiment, fis 1. In another embodiment, f is 0.

In one embodiment, a is 1, b is 1, c is 1, and f is 0.

a) Therapeutic Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure are conjugated to an effector moiety comprising a therapeuticagent, e.g. a drug moiety (or prodrug thereof) or radiolabeled compound.In one embodiment the therapeutic agent is a cytotoxin. Exemplarycytotoxic therapeutic agents are set forth in Table 1 herein.

TABLE 1 Exemplary cytotoxic therapeutic agents

  R₁ = alkyl, aryl, alkoxy, aryloxy, R₂, R₃ = alkyl, aryl

Further exemplary drug moieties include anti-inflammatory, anti-cancer,anti-infective (e.g., anti-fungal, antibacterial, anti-parasitic,anti-viral, etc.), and anesthetic therapeutic agents. In a furtherembodiment, the drug moiety is an anti-cancer agent. Exemplaryanti-cancer agents include, but are not limited to, cytostatics, enzymeinhibitors, gene regulators, cytotoxic nucleosides, tubulin bindingagents or tubulin inhibitors, proteasome inhibitors, hormones andhormone antagonists, anti-angiogenesis agents, and the like. Exemplarycytostatic anti-cancer agents include alkylating agents such as theanthracycline family of drugs (e.g. adriamycin, carminomycin,cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin,streptonigrin, porfiromycin, anthracenediones, and aziridines). Othercytostatic anti-cancer agents include DNA synthesis inhibitors (e.g.,methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside,5-fluoro-5′-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea,actinomycin-D, and mitomycin C), DNA-intercalators or cross-linkers(e.g., bleomycin, carboplatin, carmustine, chlorambucil,cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin),melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcriptionregulators (e.g., actinomycin D, daunorubicin, doxorubicin,homoharringtonine, and idarubicin). Other exemplary cytostatic agentsthat are compatible with the present disclosure include ansamycinbenzoquinones, quinonoid derivatives (e.g. quinolones, genistein,bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone,diaziquone, carbazilquinone, indoloquinone EO9,diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, andnitrosourea compounds (e.g. carmustine, lomustine, semustine).

Exemplary cytotoxic nucleoside anti-cancer agents include, but are notlimited to: adenosine arabinoside, cytarabine, cytosine arabinoside,5-fluorouracil, fludarabine, floxuridine, ftorafur, and6-mercaptopurine. Exemplary anti-cancer tubulin binding agents include,but are not limited to: taxoids (e.g. paclitaxel, docetaxel, taxane),nocodazole, rhizoxin, dolastatins (e.g. Dolastatin-10, -11, or -15),colchicine and colchicinoids (e.g. ZD6126), combretastatins (e.g.Combretastatin A-4, AVE-6032), and vinca alkaloids (e.g. vinblastine,vincristine, vindesine, and vinorelbine (navelbine)). Exemplaryanti-cancer hormones and hormone antagonists include, but are notlimited to: corticosteroids (e.g. prednisone), progestins (e.g.hydroxyprogesterone or medroprogesterone), estrogens, (e.g.diethylstilbestrol), antiestrogens (e.g. tamoxifen), androgens (e.g.testosterone), aromatase inhibitors (e.g. aminogluthetimide),17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide,apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid,leuprolide (leuprorelin), luteinizing hormone-releasing hormone,pifithrin-a, rapamycin, sex hormone-binding globulin, and thapsigargin.Exemplary anti-cancer, anti-angiogenesis compounds include, but are notlimited to: Angiostatin Kl-3, DL-a-difluoromethyl-ornithine, endostatin,fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

Exemplary anti-cancer enzyme inhibitors include, but are not limited to:S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-diCH1orobenz-imidazole1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin,2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin,trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.

Exemplary anti-cancer gene regulators include, but are not limited to:5-aza-2′-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3),4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal(vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoicacid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, andtroglitazone.

Other classes of anti-cancer agents include, but are not limited to: thepteridine family of drugs, diynenes, and the podophyllotoxins.Particularly useful members of those classes include, for example,methopterin, podophyllotoxin, or podophyllotoxin derivatives such asetoposide or etoposide phosphate, leurosidine, vindesine, leurosine andthe like.

Still other anti-cancer agents that are compatible with the teachingsherein include auristatins (e.g. auristatin E and monomethylauristan E),geldanamycin, calicheamicin, gramicidin D, maytansanoids (e.g.maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B,ethidium bromide, emetine, tenoposide, colchicin, dihydroxyanthracindione, mitoxantrone, procaine, tetracaine, lidocaine,propranolol, puromycin, and analogs or homologs thereof.

Still other anti-cancer agents that are compatible with the teachingsherein include tomaymycin derivatives, maytansine derivatives,cryptophycine derivatives, anthracycline derivatives, bisphosphonatederivatives, leptomycin derivatives, streptonigrin derivatives,auristatine derivatives, and duocarmycin derivatives.

Another class of compatible anti-cancer agents that may be used as drugmoieties are radiosensitizing drugs that may be effectively directed totumor or immunoreactive cells. Such drug moeities enhance thesensitivity to ionizing radiation, thereby increasing the efficacy ofradiotherapy. Not to be limited by theory, but an antibody modified witha radiosensitizing drug moiety and internalized by the tumor cell woulddeliver the radiosensitizer nearer the nucleus where radiosensitizationwould be maximal. Antibodies which lose the radiosensitizer moiety wouldbe cleared quickly from the blood, localizing the remainingradiosensitization agent in the target tumor and providing minimaluptake in normal tissues. After clearance from the blood, adjunctradiotherapy could be administered by external beam radiation directedspecifically to the tumor, radioactivity directly implanted in thetumor, or systemic radioimmunotherapy with the same modified antibody.

In one embodiment, the therapeutic agent comprises radionuclides orradiolabels with high-energy ionizing radiation that are capable ofcausing multiple strand breaks in nuclear DNA, leading to cell death.Exemplary high-energy radionuclides include: ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I,¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷¹Lu, ¹⁸⁶Re and ¹⁸⁸Re. Theseisotopes typically produce high energy α- or β-particles which have ashort path length. Such radionuclides kill cells to which they are inclose proximity, for example neoplastic cells to which the conjugate hasattached or has entered. They have little or no effect on non-localizedcells and are essentially non-immunogenic. Alternatively, high-energyisotopes may be generated by thermal irradiation of an otherwise stableisotope, for example as in boron neutron-capture therapy (Guan et al.,PNAS, 95: 13206-10, 1998).

In one embodiment, the therapeutic agent is selected from MMAE, MMAF,and PEGS-Do110.

Exemplary therapeutic effector moieties include the structures:

In one embodiment, the effector moiety is selected from:

In certain embodiments, the effector moiety contains more than onetherapeutic agent. These multiple therapeutic agents can be the same ordifferent.

b) Diagnostic Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure are conjugated to an effector moiety comprising a diagnosticagent. In one embodiment, the diagnostic agent is a detectable smallmolecule label e.g. biotin, fluorophores, chromophores, spin resonanceprobes, imaging agents, or radiolabels. Exemplary fluorophores includefluorescent dyes (e.g. fluorescein, rhodamine, and the like) and otherluminescent molecules (e.g. luminal). A fluorophore may beenvironmentally-sensitive such that its fluorescence changes if it islocated close to one or more residues in the modified bindingpolypeptide that undergo structural changes upon binding a substrate(e.g. dansyl probes). Exemplary radiolabels include small moleculescontaining atoms with one or more low sensitivity nuclei (¹³C, ¹⁵N, ²H,¹²⁵I, ¹²⁴I, ¹²³I, ⁹⁹Tc, ⁴³K, ⁵²Fe, ⁶⁴Cu, ⁶⁸Ga, ¹¹¹In and the like). Theradionuclide can be, for example, a gamma, photon, or positron-emittingradionuclide with a half-life suitable to permit activity or detectionafter the elapsed time between administration and localization to theimaging site.

In one embodiment, the diagnostic agent is a polypeptide. Exemplarydiagnostic polypeptides include enzymes with fluorogenic or chromogenicactivity, e.g. the ability to cleave a substrate which forms afluorophore or chromophore as a product (i.e. reporter proteins such asluciferase). Other diagnostic proteins may have intrinsic fluorogenic orchromogenic activity (e.g., green, red, and yellow fluorescentbioluminescent aequorin proteins from bioluminescent marine organisms)or they may comprise a protein containing one or more low-energyradioactive nuclei (¹³C, ¹⁵N, ²H, ¹²⁵I, ¹²⁴I, ¹²³I, ⁹⁹Tc, ⁴³K, ⁵²Fe,⁶⁴Cu, ⁶⁸Ga, ¹¹¹In and the like).

With respect to the use of radiolabeled conjugates in conjunction withthe present disclosure, binding polypeptides of the current disclosuremay be directly labeled (such as through iodination) or may be labeledindirectly through the use of a chelating agent. As used herein, thephrases “indirect labeling” and “indirect labeling approach” both meanthat a chelating agent is covalently attached to a binding polypeptideand at least one radionuclide is associated with the chelating agent.Such chelating agents are typically referred to as bifunctionalchelating agents as they bind both the polypeptide and the radioisotope.Exemplary chelating agents comprise1-isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid(“MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid(“CHX-DTPA”) derivatives. Other chelating agents comprise P-DOTA andEDTA derivatives. Particularly radionuclides for indirect labelinginclude ¹¹¹In and ⁹⁰Y. Most imaging studies utilize 5 mCi ¹¹¹In-labeledantibody, because this dose is both safe and has increased imagingefficiency compared with lower doses, with optimal imaging occurring atthree to six days after antibody administration. See, for example,Murray, (1985), J. Nuc. Med. 26: 3328 and Carraguillo et al, (1985), J.Nuc. Med. 26: 67. The radionuclide for direct labeling can be, forexample, ¹³¹I. Those skilled in the art will appreciate thatnon-radioactive conjugates may also be assembled depending on theselected agent to be conjugated.

In certain embodiments, the diagnostic effector moiety is a FRET(Fluorescence Resonance Energy Transfer) probe. FRET has been used for avariety of diagnostic applications including cancer diagnostics. A FRETprobe may include a cleavable linker (enzyme sensitive or pH linker)connecting the donor and acceptor moieties of the FRET probe, whereincleavage results in enhanced fluorescence (including near Infrared)(see, e.g., A. Cobos-Correa et. al. Membrane-bound FRET probe visualizesMMP12 activity in pulmonary inflammation, Nature Chemical Biology(2009), 5(9), 628-63; S. Gehrig et. al. Spatially Resolved Monitoring ofNeutrophil Elastase Activity with Ratiometric Fluorescent Reporters(2012) Angew. Chem. Int. Ed., 51, 6258-6261).

In one embodiment, the effector moiety is selected from:

c) Functionalized Effector Moieties

In certain embodiments, effector moieties may be functionalized tocontain additional groups in addition to the effector moiety itself. Forexample, the effector moiety may contain cleavable linkers which releasethe effector moiety from the binding polypeptide under particularconditions. In exemplary embodiments, the effector moiety may include alinker that is cleavable by cellular enzymes and/or is pH sensitive.Additionally or alternatively, the effector moiety may contain adisulfide bond that cleaved by intracellular glutathione upon uptakeinto the cell. Exemplary disulfide and pH sensitive linkers are providedbelow:

In yet other embodiments, the effector moiety may include hydrophilicand biocompatible moieties such as poly(glycine), poly(oxazoline), orPEG moieties. Exemplary structures (“Y”) are provided below:

In certain embodiments, the effector moiety contains an aminooxy groupwhich facilitates conjugation to a binding polypeptide via a stableoxime linkage.

In other embodiments, the effector moiety contains a hydrazide and/orN-alkylated hydrazine group to facilitate conjugation to a bindingpolypeptide via a stable hydrazone linkage. Exemplary effector moietiescontaining aminooxy groups are set forth in Table 14 herein.

TABLE 14 Exemplary hydrazine and/or hydrazide effector moieties

d) Targeting Moieties

In certain embodiments, effector moieties comprise targeting moietiesthat specifically bind to one or more target molecules. Any type oftargeting moiety can be employed including, without limitation,proteins, nucleic acids, lipids, carbohydrates (e.g., glycans), andcombinations thereof (e.g., glycoproteins, glycopeptides, andglycolipids). In certain embodiments, the targeting moiety is acarbohydrate or glycopeptide. In one embodiment, the targeting moiety isa trivalent glycopeptide (e.g. a trivalent GalNAc glycan containingglycopeptide or a trivalent galactose containing glycopeptide). In aspecific embodiment, the trivalent galactose containing polypeptide islactose3-Cys3Gly4. In certain embodiments, the targeting moiety is aglycan. Targeting moieties can be naturally or non-naturally occurringmolecules. Targeting moieties suitable for conjugation may include thosecontaining aminooxy linkers (see, e.g., FIGS. 45 and 46).

The targeting moieties described in the present invention may bind toany type of cell, including animal (e.g., mammalian), plant, or insectcells either in vitro or in vivo, without limitation. The cells may beof endodermal, mesodermal, or ectodermal origins, and may include anycell type. In certain embodiments, the targeting moiety binds to a cell,e.g., a mammalian cell, a facilitates delivery of a binding polypeptideto the targeted cell, e.g., to improve cell-targeting and/or uptake.Exemplary target cells include, without limitation, immune cells (e.g.,lymphocytes such as B cells, T cells, natural killer (NK) cells,basophils, macrophages, or dendritic cells), liver cells (e.g.,hepatocytes or non-parenchymal cells such as liver sinusoidalendothelial cells, Kupffer cells, or hepatic stellate cells), tumorcells (e.g., any malignant or benign cell including hepatoma cells, lungcancer cells, sarcoma cells, leukemia cells, or lymphoma cells),vascular cells (e.g., aortic endothelial cells or pulmonary arteryendothelial cells), epithelial cells (e.g., simple squamous epithelialcells, simple columnar epithelial cells, pseudostratified columnarepithelial cells, or stratified squamous epithelial cells), ormesenchymal cells (e.g., cells of the lymphatic and circulatory systems,bone, and cartilage cells).

In one embodiment, the binding polypeptide is internalized by the cell.In another embodiment, the amount of the binding polypeptideinternalized by the cell is greater than the amount of a referencebinding polypeptide lacking a targeting moiety internalized by the cell.

In one embodiment, the targeting moiety binds to a receptor on thetarget cell. For example, the targeting moiety may comprise a mannose 6phosphate moiety that binds to a mannose 6 phosphate receptor on thecell. In other exemplary embodiments, the targeting moiety binds to aSiglec on a target cell. Exemplary Siglecs include sialoadhesin(Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3), MAG (Siglec-4), Siglec-5,Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12,Siglec-14, or Siglec-15. In yet other embodiments, the targeting moietycomprises an α2,3-, α2,6-, or α2,8-linked sialic acid residue. In afurther embodiment, the targeting moiety comprises an α2,3-siallylactosemoiety or an α2,6-siallylactose moiety. Other exemplary receptorsinclude lectin receptors, including but not limited to C-type lectinreceptors, galectins, and L-type lectin receptors. Exemplary lectinreceptors include: TDEC-205, macrophage mannose receptor (MMR),Dectin-1, Dectin-2, macrophage-inducible C-type lectin (Mincle),dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN, CD209), DCNK lectin group receptor-1 (DNGR-1), Langerin (CD207), CD169, alectican, an asialoglycoprotein receptor, DCIR, MGL, a DC receptor, acollectin, a selectin, an NK-cell receptor, a multi-CTLD endocyticreceptor, a Reg group (type VII) lectin, chondrolectin, tetranectin,polycystin, attractin (ATRN), eosinophil major basic protein (EMBP),DGCR2, Thrombomodulin, Bimlec, SEEC, and CBCP/Frem1/QBRICK.

The binding polypeptides of the present invention may be used to removetoxic compounds and harmful substances into liver in multiple diseasesby targeting carbohydrate receptors (e.g., mannose 6-phosphate receptor,mannose receptor, and asialoglycoprotein receptor). Please see: Ganesan,L. P. et al: Rapid and Efficient Clearance of Blood-borne Virus by LiverSinusoidal Endothelium. PLoS Pathogens 2011, 9: 1; and Monnier, V. M. etal: Glucosepane: a poorly understood advanced glycation end product ofgrowing importance for diabetes and its complications. Clin Chem Lab Med2014; 52: 21.

The binding polypeptides of the present invention may also be used totarget tumor cells through targeting different cell receptors including,but not limited to: carbohydrate receptors, Asialoglycoprotein receptor,and Siglecs. Please see: Chen, W. C. et al: In vivo targeting of B-celllymphoma with glycan ligands of CD22. Blood 2010, 115: 4778; Chen, W. C.et al: Targeting B lymphoma with nanoparticles bearing glycan ligands ofCD22. Leuk Lymphoma 2012, 53: 208; Hatakeyama, S. et al: Targeted drugdelivery to tumor vasculature by a carbohydrate mimetic peptide. PNAS,2011, 108: 19587; Hong, F. et al: β-Glucan Functions as an Adjuvant forMonoclonal Antibody Immunotherapy by Recruiting Tumoricidal Granulocytesas Killer Cells. Cancer Res. 2003, 23: 9023; Kawasakia, N. et al:Targeted delivery of lipid antigen to macrophages via theCD169/sialoadhesin endocytic pathway induces robust invariant naturalkiller T cell activation. PNAS 2013, 110: 7826; and Medina, S. H. et al:N-acetylgalactosamine-functionalized dendrimers as hepatic cancercell-targeted carriers. Biomaterials 2011, 32: 4118.

The binding peptides of the present invention may also be used toregulate immune response through various receptors including, but notlimited to, carbohydrate receptors, DC-SIGN, or Siglecs. Please see:Anthony, R. M. et al: Recapitulation of IVIG Anti-Inflammatory Activitywith a Recombinant IgG Fc. Science 2008, 320: 373; Anthony, R. M. et al:Identification of a receptor required for the anti-inflammatory activityof IVIG. PNAS 2008, 105: 19571; Kaneko, Y. et al: Anti-InflammatoryActivity of Immunoglobulin G Resulting from Fc Sialylation. Science2006, 313: 670; and Mattner, J. et al: Exogenous and endogenousglycolipid antigens activate NKT cells during microbial infections.Nature 2005, 434: 525.

In one embodiment, the targeting moiety is a glycopeptide. In a furtherembodiment, the targeting moiety is a tri-galactosylated glycopeptide,e.g., lactose₃-Cys₃Gly₄ (shown in Formula V, below):

e) PEG Moieties

In other aspects, the effector moiety is a moiety comprisingpoly(ethylene glycol) (PEG, PEO, or POE). PEG is an oligomer or polymerof ethylene oxide and has the chemical structure H—(O—CH₂—CH₂)n-OHwherein the element in parentheses is repeated. PEGylation (orpegylation) is a process in which PEG polymer chains are attached toanother molecule (e.g., a binding polypeptide), which is then describedas PEGylated (or pegylated). PEGylation can serve to reduceimmunogenicity and antigenicity as well as to increase the hydrodynamicsize (size in solution) of the molecule it is attached to, reducingrenal clearance and prolonging circulation time. PEGylation can alsomake molecules more water soluble. In one embodiment of the presentinvention, the PEG moiety may comprise mono-PEG, bi-PEG, or tri-PEG. Inanother embodiment, the PEG moiety comprises 3 to 3.5 PEG.

VII. Conjugation of Effector Moieties to Binding Polypeptides

In certain embodiments, effector moieties are conjugated (eitherdirectly or through a linker moiety) to an oxidized glycan (e.g., anoxidized N-linked glycan) of an altered binding polypeptide, (e.g., anengineered glycan at N114 of an antibody CH1 domain or a native glycanat N297 of an antibody F domain). The term “oxidized glycan” means thatan alcohol substituent on the glycan has been oxidized, providing acarbonyl substituent. The carbonyl substituent can react with suitablenitrogen nucleophile to form a carbon-nitrogen double bond. For example,reaction of the carbonyl group with an aminooxy group or hydrazine groupwould form an oxime or hydrazine, respectively. In one embodiment, thecarbonyl substituent is an aldehyde. Suitable oxidized glycans includeoxidized galactose and oxidized sialic acid.

In one embodiment, the modified polypeptide of Formula (II) may be ofFormula (II):Ab(Gal-C(O)H)_(x)(Gal-Sia-C(O)H)_(y)   Formula (II),wherein

A) Ab is an antibody or other binding polypeptide as defined herein;

B) Gal is a component derived from galactose;

C) Sia is a component derived from sialic acid;

D) x is 0 to 5; and

E) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

Any art recognized chemistry can be employed to conjugate an effectormoiety (e.g., an effector moiety comprising a linker moiety) to a glycan(see e.g., Hermanson, G. T., Bioconjugate Techniques. Academic Press(1996), which is incorporated herein ion its entirety). In certainembodiments, a saccharide residue (e.g., a sialic acid or galactoseresidue) of the glycan is first oxidized (e.g., using sodium periodatetreatment of sialic acid or galactose oxidase treatment of galactose) togenerate a reactive aldehyde group. This aldehyde group is reacted witheffector moiety an aminooxy group or hydrazine group to form an oxime orhydrazone linker, respectively. Exemplary methods employing this generalreaction scheme are set forth in Examples 10 to 15.

In certain embodiments, the native or engineered glycans of a bindingpolypeptide are first pre-treated with glycosyltransferase enzymes invitro to provide a terminal saccharide residue that is suitablyreactive. For example, sialylation may be achieved first using acombination of galactosyltransferase (Gal T) and sialyltransferase (SialT). In certain embodiments, biantennary glycans that lack galactose (G0For G0) or that contain only one galactose (G1F or G1) can be convertedto higher-order galactosylated or sialylated structures suitable forconjugation (G1F, G1, G2F, G2, G1S1F, G1S1, G2S1F, G2S1, G2S2F, orG2S2).

An exemplary conjugation scheme for producing sialylated glycoconjugatesis shown in FIG. 30C. An exemplary conjugation scheme for producingsialylated glycoconjugates is shown in FIG. 30B. Sialic acid residuesare introduced enzymatically and site specifically into the glycan of anantibody (e.g., a native glycan at Asn-297) using a combination ofgalactosyltransferase (Gal T) and sialyltransferase (Sial T). Introducedsialic acid residues are subsequently oxidized with a low concentrationof sodium periodate to yield reactive sialic acid aldehydes suitablyreactive with linkers (e.g., aminooxy linkers) to generateantibody-effector moiety conjugates (e.g., oxime-linkedantibody-effector moiety conjugates). By controlling the number ofglycan and the number of sialic residues with in vitro remodeling, theskilled artisan may have precise control over the drug-antibody ratio(DAR) of the antibody-effector moiety conjugates. For example, if ˜1sialic acid is added onto a single biantennary glycan (A1F) in each ofheavy chain, an antibody or binding polypeptide with a DAR of 2 can behomogeneously obtained.

Oxidation and Oxidation Agents

Oxidation can have adverse effects on the integrity of an antibody, boththrough the oxidation of monosaccharides and through the oxidation ofamino acids. The oxidation of methionine residues, including Met-252 andMet-428 (located in Fc CH3 region, proximal to FcRn binding site) isknown to affect FcRn binding, which is critical for prolonging antibodyserum half-life (Wang, W., et al. (2011) Impact of methionine oxidationin human IgG1 Fc on serum half-life of monoclonal antibodies. MolImmunol 48, 860-6). Accordingly, attempts have previously been made toreduce the amount of oxidizing agents (e.g. periodate oxidase orgalactose oxidase) used to treat binding proteins comprising glycans inorder to create oxidized groups for conjugation to effector moieties.

The method of the present invention uses CMP-sialic acid derivativescomprising reactive moieties (including, but not limited to, an aldehydemoiety, an alkyne, an aminooxy moiety, an azide, a hydrazine, a ketomoiety, or a thiol), which may be reacted with a binding polypeptide inorder to form a sialic acid derivative-conjugated binding protein. Thesesialic acid derivative-conjugated binding proteins can then be coupledto different effector moieties without treatment with an oxidizingagent.

Imine Chemistry

In some embodiments, the CMP-sialic acid derivative comprises a reactivemoiety including an aldehyde, a keto, a hydrazine, or a hydrazonemoiety. In some embodiments, the reactive moiety is a terminal reactivemoiety including, but not limited to, a terminal aldehyde or a terminalketo moiety. In example embodiments, the CMP-sialic acid derivative hasone of the following structural formulas:

wherein R₂ includes, but is not limited to, CH₃, CH₂CH₂(C═O)CH₃, CH₂OH,OH or H. In some embodiments, the CMP-sialic acid derivative comprisinga terminal aldehyde moiety includes, but is not limited to, thefollowing structural formulas:

In some embodiments, the sialic acid derivative-conjugated bindingpolypeptide comprises a reactive moiety including an aldehyde, a keto, ahydrazine, or a hydrazone moiety. In example embodiments, the sialicacid derivative-conjugated binding polypeptide may be represented by thefollowing:

wherein X represents the remainder of the sialic acidderivative-conjugated binding polypeptide (i.e., other than the reactivemoiety).

In some embodiments, the effector or targeting moiety comprises areactive moiety including an aldehyde, a keto, a hydrazine, or ahydrazone moiety. In example embodiments, the effector or targetingmoiety may be represented by the following:

wherein X represents the remainder of the effector or targeting moiety(i.e., other than the reactive moiety).

In some embodiments, a targeting or effector moiety conjugated bindingpolypeptide comprises an imine. In example embodiments, a type of imineincludes, but is not limited to, an aldimine, a hydroxylamine, ahydrazone, a ketamine, or an oxime. For example, see FIG. 3 (A-C) forimine formation. In example embodiments, the imine of a targeting oreffector moiety conjugated binding polypeptide is formed by reacting asialic acid derivative-conjugated binding polypeptide comprising analdehyde or a keto moiety with an effector or targeting moietycomprising an aminooxy moiety or is bound to a moiety comprising anaminooxy derivative or a hydrazine moiety. In example embodiments, theimine of the targeting or effector moiety conjugated binding polypeptideis formed by reacting a sialic acid derivative-conjugated bindingpolypeptide comprising an aminooxy moiety or is bound to a moietycomprising an aminooxy derivative or a hydrazine moiety with an effectoror targeting moiety comprising an aldehyde or a keto moiety.

Click Chemistry

In some embodiments, the CMP-sialic acid derivative comprises a terminalazide moiety. For example, the CMP-sialic acid derivative may be aCMP-sialic acid C5 azide derivative or a sialic acid C5 azide. Inexample embodiments, the CMP-sialic acid derivative has the followingstructural formula:

wherein R₁ is a reactive moiety including, but not limited to,NH(C═O)CH₃, NH(C═O)C₄H₇O, NH(C═O)CH₂OH, NH(C═O)CH₂N₃, NH(C═O)SH, OH orN₃. In some embodiments, the CMP-sialic acid derivative has a structuralformula selected from the following:

In some embodiments, the CMP-sialic acid derivative comprises a moietycomprising an alkyne or is bound to a moiety comprising an alkyne. Insome embodiments, the CMP-sialic acid derivative comprises or is boundto a cyclooctyne including, but not limited to, an azadibenzocyclooctyne(DBCO, ADIBO, DIBAC) moiety, a monofluorinated cyclooctyne, or adifluorinated cyclooctyne.

In some embodiments, the sialic acid derivative-conjugated bindingpolypeptide comprises a terminal azide moiety. For example, the sialicacid derivative-conjugated binding polypeptide may be a sialic acid C5azide derivative-conjugated binding polypeptide. In one exemplaryembodiment, the sialic acid derivative-conjugated binding polypeptidehas the structural formulas represented in FIG. 95. In another exampleembodiment, the sialic acid derivative-conjugated binding polypeptidehas one of the following structural formulas

wherein X represents the remainder of the sialicacid-derivative-conjugated binding polypeptide (i.e., other than theterminal azide moiety).

In some embodiments, the sialic acid derivative-conjugated bindingpolypeptide comprises a moiety comprising an alkyne or is bound to amoiety comprising an alkyne. In some embodiments, the sialic acidderivative-conjugated binding polypeptide comprises or is bound to acyclooctyne including, but not limited to, an azadibenzocyclooctyne(e.g., DBCO, ADIBO, DIBAC) moiety, a monoflorimated cyclooctyne, or adifluorinated cyclooctyne.

In some embodiments, the effector or targeting moiety comprises analkyne or is bound to a moiety comprising an alkyne. In someembodiments, the effector or targeting moiety comprises or is bound to acyclooctyne including, but not limited to, an azadibenzocyclooctyne(e.g., DBCO, ADIBO, DIBAC) moiety, a monoflorimated cyclooctyne, or adifluorinated cyclooctyne. In example embodiments, the effector ortargeting moiety is bound to a moiety comprising an alkyne and can berepresented by the following structural formula:

In some embodiments, the effector or targeting moiety comprises aterminal azide moiety.

In some embodiments, a targeting or effector moiety conjugated bindingpolypeptide comprises a triazole ring. In example embodiments, thetriazole ring of a targeting or effector moiety conjugated bindingpolypeptide is formed by reacting a sialic acid derivative-conjugatedbinding polypeptide comprising a terminal azide moiety with an effectoror targeting moiety comprising an alkyne or bound to a moiety comprisingan alkyne using click chemistry. In example embodiments, the triazolering of the targeting or effector moiety conjugated binding polypeptideis formed by reacting a sialic acid derivative-conjugated bindingpolypeptide comprising an alkyne or bound to a moiety comprising analkyne with an effector or targeting moiety comprising a terminal azidemoiety using click chemistry. In some embodiments, the click chemistryreaction to form the targeting or effector moiety conjugated bindingpolypeptide occurs at ambient temperatures. In some embodiments, theclick chemistry reaction to form a targeting or effector moietyconjugated binding polypeptide occurs in the presence of a metalcatalyst, for example, a copper(I)-catalyzed azide-alkyne cycloaddition.In some embodiments, the click chemistry reaction to form a targeting oreffector moiety conjugated binding polypeptide is performed in theabsence of copper.

In some embodiments, the mechanism of a click chemistry reaction to forma targeting or effector moiety conjugated binding polypeptide includes,but is not limited to, a copper(I)-catalyzed [3+2] azide-alkynecycloaddition, a strain-promoted [3+2] azide-alkyne cycloaddition, a[3+2] Huisgen cycloaddition between an azide moiety and an activatedalkyne, a [3+2] cycloaddition between an azide moiety and anelectron-deficient alkyne, a [3+2] cycloaddition between an azide and anaryne, a Diels-Alder retro-[4+2] cycloaddition between a tetrazine andan alkene, or a radical addition between a thiol and an alkene.

Thioether Chemistry

In some embodiments, the CMP-sialic acid derivative or sialic acidderivative comprises a reactive moiety including a thiol or a maleimidemoiety. In some embodiments, the CMP-sialic acid derivative or sialicacid derivative comprises a terminal thiol. In example embodiments, theCMP-sialic acid derivative comprising a terminal thiol includes, but isnot limited to a structural formula of:

In some embodiments, the sialic acid derivative-conjugated bindingpolypeptide comprises a reactive moiety including a thiol or a maleimidemoiety. In some embodiments, the sialic acid derivative-conjugatedbinding polypeptide comprises a terminal thiol moiety. In exampleembodiments, the sialic acid derivative-conjugated binding polypeptidemay be represented by, but is not limited to, the following:

wherein X is the remainder of the sialic acid derivative-conjugatedbinding polypeptide (i.e., other than the thiol or maleimide moiety).

In some embodiments, the effector or targeting moiety comprises areactive moiety including a thiol or a maleimide moiety. In someembodiments, the effector or targeting moiety comprises a terminal thiolmoiety. In some embodiments, the effector or targeting moiety comprisesa terminal maleimide moiety. For example, the effector or targetingmoiety comprising a maleimide moiety includes, but is not limited to,bis-mannose-6-phosphate hexamannose maleimide, or lactose maleimide.

In example embodiments, the effector or targeting moiety may berepresented by, but is not limited to, the following:

wherein X represents the remainder of the effector or targeting moiety.

In some embodiments, a targeting or effector moiety conjugated bindingpolypeptide comprises a thioether. In example embodiments, the thioetherof a targeting or effector moiety conjugated binding polypeptide isformed by reacting a sialic acid derivative-conjugated bindingpolypeptide comprising a thiol moiety with an effector or targetingmoiety comprising a maleimide moiety. In example embodiments, the imineof the targeting or effector moiety conjugated binding polypeptide isformed by reacting a sialic acid derivative-conjugated bindingpolypeptide comprising a maleimide moiety with an effector or targetingmoiety comprising a thiol moiety.

VIII. Modified Binding Polypeptides

In certain embodiments, the invention provides modified polypeptideswhich are the product of the conjugating effector moieties areconjugated (either directly or through a linker moiety) to an oxidizedglycan (e.g., an oxidized N-linked glycan) of an altered bindingpolypeptide (e.g., an engineered glycan at N114 of an antibody CH1domain or a native glycan at N297 of an antibody F domain).

In one embodiment, the binding polypeptide can be of Formula (III):Ab(Gal-C(H)═N-Q-CON—X)_(x)(Gal-Sia-C(H)═N-Q-CON—X)_(y)   Formula (III),wherein:

A) Ab is an antibody as defined herein;

B) Q is NH or O;

C) CON is a connector moiety as defined herein; and

D) X is a targeting moiety as defined herein;

E) Gal is a component derived from galactose;

F) Sia is a component derived from sialic acid;

G) x is 0 to 5; and

H) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

In one embodiment, the binding polypeptide can be of Formula (III) canbe of Formula (IIIa):Ab(Gal-C(H)═N-Q-CH₂—C(O)—Z—X)_(x)(Gal-Sia-C(H)═N-Q-CH₂—C(O)—Z—X)_(y)  Formula (IIIa),wherein:

A) Ab is an antibody;

B) Q is NH or O;

C) Z is Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—, wherein

-   -   i. Cys is a component derived cysteinamide;    -   ii. MC is a component derived from maleimide;    -   iii. VC is a component derived from valine coupled with        citruline;    -   iv. PABC is a component derived from 4-aminobenzyl carbamate;    -   v. X is an effector moiety (e.g., a targeting moiety as defined        herein);    -   vi. a is 0 or 1;    -   vii. b is 0 or 1;    -   viii. c is 0 or 1; and    -   ix. f is 0 or 1;

D) X is a therapeutic agent as defined herein;

E) Gal is a component derived from galactose;

F) Sia is a component derived from sialic acid;

G) x is 0 to 5; and

H) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

It is to be understood that the Formula (III) is not intended to implythat the antibody, the Gal substituent, and the Gal-Sia substituent areconnected in a chain-like manner. Rather, when such substituents arepresent, the antibody is connected directly connected to eachsubstituent. For example, a binding polypeptide of Formula (III) inwhich x is 1 and y is 2 could have the arrangement shown below:

The CON substituent in Formula (III) and components therein are asdescribed with regard to Formula (I) for effector moieties.

In one embodiment, Q is NH. In another embodiment, Q is O.

In one embodiment, x is 0.

The antibody Ab of Formula (III) may be any suitable antibody asdescribed herein.

In one embodiment, there is provided a method for preparing the bindingpolypeptide of Formula (III), the method comprising reacting an effectormoiety of Formula (I):NH₂-Q-CON—X   Formula (I),wherein:

A) Q is NH or O;

B) CON is a connector moiety; and

C) X is an effector moiety (e.g., a targeting moiety as defined herein),

with a modified antibody of Formula (II)Ab(OXG)_(r)   Formula (II)wherein

A) OXG is an oxidized glycan; and

B) r is selected from 0 to 4;

In one embodiment, there is provided a method for preparing the bindingpolypeptide of Formula (III), the method comprising reacting an effectormoiety of Formula (I):NH₂-Q-CON—X   Formula (I),wherein:

A) Q is NH or O;

B) CON is a connector moiety; and

C) X is an effector moiety (e.g., a targeting moiety as defined herein),

with a modified antibody of Formula (IIa)Ab(Gal-C(O)H)_(x)(Gal-Sia-C(O)H)_(y)   Formula (IIa),wherein

A) Ab is an antibody as described herein;

B) Gal is a component derived from galactose;

C) Sia is a component derived from sialic acid;

D) x is 0 to 5; and

E) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

IX. Methods of Treatment with Modified Antibodies

In one aspect, the invention provides methods of treating or diagnosinga patient in need thereof comprising administering an effective amount abinding polypeptide disclosed herein. In some embodiments, the presentinvention includes kits and methods for the diagnosis and/or treatmentof disorders, e.g., neoplastic disorders in a mammalian subject in needof such treatment. In some embodiments, the subject is a human.

The binding polypeptides of the current disclosure are useful in anumber of different applications. For example, in one embodiment, thesubject binding polypeptide s are useful for reducing or eliminatingcells bearing an epitope recognized by the binding domain of the bindingpolypeptide. In another embodiment, the subject binding polypeptides areeffective in reducing the concentration of or eliminating solubleantigen in the circulation. In one embodiment, the binding polypeptidesmay reduce tumor size, inhibit tumor growth and/or prolong the survivaltime of tumor-bearing animals. Accordingly, this disclosure also relatesto a method of treating tumors in a human or other animal byadministering to such human or animal an effective, non-toxic amount ofmodified antibody. One skilled in the art would be able, by routineexperimentation, to determine what an effective, non-toxic amount ofmodified binding polypeptide would be for the purpose of treatingmalignancies. For example, a therapeutically active amount of a modifiedantibody or fragments thereof may vary according to factors such as thedisease stage (e.g., stage I versus stage IV), age, sex, medicalcomplications (e.g., immunosuppressed conditions or diseases) and weightof the subject, and the ability of the modified antibody to elicit adesired response in the subject. The dosage regimen may be adjusted toprovide the optimum therapeutic response. For example, several divideddoses may be administered daily, or the dose may be proportionallyreduced as indicated by the exigencies of the therapeutic situation.

In general, the compositions provided in the current disclosure may beused to prophylactically or therapeutically treat any neoplasmcomprising an antigenic marker that allows for the targeting of thecancerous cells by the modified antibody.

X. Methods of Administering Modified Antibodies or Fragments Thereof

Methods of preparing and administering binding polypeptides of thecurrent disclosure to a subject are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe binding polypeptides of the current disclosure may be oral,parenteral, by inhalation or topical. The term parenteral as used hereinincludes intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal or vaginal administration. While all these forms ofadministration are clearly contemplated as being within the scope of thecurrent disclosure, a form for administration would be a solution forinjection, in particular for intravenous or intraarterial injection ordrip. Usually, a suitable pharmaceutical composition for injection maycomprise a buffer (e.g. acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. humanalbumin), etc. However, in other methods compatible with the teachingsherein, the modified antibodies can be delivered directly to the site ofthe adverse cellular population thereby increasing the exposure of thediseased tissue to the therapeutic agent.

In one embodiment, the binding polypeptide that is administered is abinding polypeptide of Formula (III):Ab(Gal-C(H)═N-Q-CON—X)_(x)(Gal-Sia-C(H)═N-Q-CON—X)_(y)   Formula (III),wherein:

A) Ab is an antibody as defined herein;

B) Q is NH or O;

C) CON is a connector moiety as defined herein; and

D) X is an effector moiety (e.g., a targeting moiety as defined herein);

E) Gal is a component derived from galactose;

F) Sia is a component derived from sialic acid;

G) x is 0 to 5; and

H) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the compositions and methods of the current disclosure,pharmaceutically acceptable carriers include, but are not limited to,0.01-0.1 M or 0.05M phosphate buffer, or 0.8% saline. Other commonparenteral vehicles include sodium phosphate solutions, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present such asfor example, antimicrobials, antioxidants, chelating agents, and inertgases and the like. More particularly, pharmaceutical compositionssuitable for injectable use include sterile aqueous solutions (wherewater soluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In suchcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage, and should also be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. Isotonicagents, for example, sugars, polyalcohols, such as mannitol, sorbitol,or sodium chloride may also be included in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., a modified binding polypeptideby itself or in combination with other active agents) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle, which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation typically includevacuum drying and freeze-drying, which yield a powder of an activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparations for injections areprocessed, filled into containers such as ampoules, bags, bottles,syringes or vials, and sealed under aseptic conditions according tomethods known in the art. Further, the preparations may be packaged andsold in the form of a kit such as those described in co-pending U.S.Ser. No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which isincorporated herein by reference. Such articles of manufacture caninclude labels or package inserts indicating that the associatedcompositions are useful for treating a subject suffering from, orpredisposed to autoimmune or neoplastic disorders.

Effective doses of the compositions of the present disclosure, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages may be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

For passive immunization with a binding polypeptide, the dosage canrange, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2mg/kg, etc.), of the host body weight. For example dosages can be 1mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg, e.g., at least 1 mg/kg. Doses intermediate in the above rangesare also intended to be within the scope of the current disclosure.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimens entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated.

Binding polypeptides of the current disclosure can be administered onmultiple occasions. Intervals between single dosages can be weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of modified binding polypeptide or antigen in thepatient. In some methods, dosage is adjusted to achieve a plasmamodified binding polypeptide concentration of 1-1000 μg/ml and in somemethods 25-300 μg/ml. Alternatively, binding polypeptides can beadministered as a sustained release formulation, in which case lessfrequent administration is required. For antibodies, dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, humanized antibodies show the longest half-life,followed by chimeric antibodies and nonhuman antibodies.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage (e.g., from about 1to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used for radioimmunoconjugates and higher doses forcytotoxin-drug modified antibodies) at relatively short intervals issometimes required until progression of the disease is reduced orterminated, or until the patient shows partial or complete ameliorationof disease symptoms. Thereafter, the patient can be administered aprophylactic regime.

Binding polypeptides of the current disclosure can optionally beadministered in combination with other agents that are effective intreating the disorder or condition in need of treatment (e.g.,prophylactic or therapeutic). Effective single treatment dosages (i.e.,therapeutically effective amounts) of ⁹⁰Y-labeled modified antibodies ofthe current disclosure range from between about 5 and about 75 mCi, suchas between about 10 and about 40 mCi. Effective single treatmentnon-marrow ablative dosages of ¹³¹I-modified antibodies range frombetween about 5 and about 70 mCi, such as between about 5 and about 40mCi. Effective single treatment ablative dosages (i.e., may requireautologous bone marrow transplantation) of ¹³¹I-labeled antibodies rangefrom between about 30 and about 600 mCi, such as between about 50 andless than about 500 mCi. In conjunction with a chimeric antibody, owingto the longer circulating half-life vis-a-vis murine antibodies, aneffective single treatment non-marrow ablative dosages of iodine-131labeled chimeric antibodies range from between about 5 and about 40 mCi,e.g., less than about 30 mCi. Imaging criteria for, e.g., the ¹¹¹Inlabel, are typically less than about 5 mCi.

While the binding polypeptides may be administered as describedimmediately above, it must be emphasized that in other embodimentsbinding polypeptides may be administered to otherwise healthy patientsas a first line therapy. In such embodiments the binding polypeptidesmay be administered to patients having normal or average red marrowreserves and/or to patients that have not, and are not, undergoing oneor more other therapies. As used herein, the administration of modifiedantibodies or fragments thereof in conjunction or combination with anadjunct therapy means the sequential, simultaneous, coextensive,concurrent, concomitant, or contemporaneous administration orapplication of the therapy and the disclosed antibodies. Those skilledin the art will appreciate that the administration or application of thevarious components of the combined therapeutic regimen may be timed toenhance the overall effectiveness of the treatment. For example,chemotherapeutic agents could be administered in standard, well knowncourses of treatment followed within a few weeks byradioimmunoconjugates of the present disclosure. Conversely, cytotoxinassociated binding polypeptides could be administered intravenouslyfollowed by tumor localized external beam radiation. In yet otherembodiments, the modified binding polypeptide may be administeredconcurrently with one or more selected chemotherapeutic agents in asingle office visit. A skilled artisan (e.g. an experienced oncologist)would be readily be able to discern effective combined therapeuticregimens without undue experimentation based on the selected adjuncttherapy and the teachings of the instant specification.

In this regard it will be appreciated that the combination of thebinding polypeptides and the chemotherapeutic agent may be administeredin any order and within any time frame that provides a therapeuticbenefit to the patient. That is, the chemotherapeutic agent and bindingpolypeptides may be administered in any order or concurrently. Inselected embodiments the binding polypeptides of the present disclosurewill be administered to patients that have previously undergonechemotherapy. In yet other embodiments, the binding polypeptides and thechemotherapeutic treatment will be administered substantiallysimultaneously or concurrently. For example, the patient may be giventhe binding polypeptides while undergoing a course of chemotherapy. Insome embodiments the modified antibody will be administered within oneyear of any chemotherapeutic agent or treatment. In other embodimentsthe binding polypeptides will be administered within 10, 8, 6, 4, or 2months of any chemotherapeutic agent or treatment. In still otherembodiments the binding polypeptide will be administered within 4, 3, 2,or 1 week(s) of any chemotherapeutic agent or treatment. In yet otherembodiments the binding polypeptides will be administered within 5, 4,3, 2, or 1 day(s) of the selected chemotherapeutic agent or treatment.It will further be appreciated that the two agents or treatments may beadministered to the patient within a matter of hours or minutes (i.e.substantially simultaneously).

It will further be appreciated that the binding polypeptides of thecurrent disclosure may be used in conjunction or combination with anychemotherapeutic agent or agents (e.g. to provide a combined therapeuticregimen) that eliminates, reduces, inhibits or controls the growth ofneoplastic cells in vivo. Exemplary chemotherapeutic agents that arecompatible with the current disclosure include alkylating agents, vincaalkaloids (e.g., vincristine and vinblastine), procarbazine,methotrexate and prednisone. The four-drug combination MOPP(mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazineand prednisone) is very effective in treating various types of lymphoma,and can be used in certain embodiments. In MOPP-resistant patients, ABVD(e.g., adriamycin, bleomycin, vinblastine and dacarbazine), ChIVPP(CH1orambucil, vinblastine, procarbazine and prednisone), CABS(lomustine, doxorubicin, bleomycin and streptozotocin), MOPP plus ABVD,MOPP plus ABV (doxorubicin, bleomycin and vinblastine) or BCVPP(carmustine, cyclophosphamide, vinblastine, procarbazine and prednisone)combinations can also be used. Arnold S. Freedman and Lee M. Nadler,Malignant Lymphomas, in HARRISON'S PRINCIPLES OF INTERNAL MEDICINE1774-1788 (Kurt J. Isselbacher et al, eds., 13th ed. 1994) and V. T.DeVita et al, (1997) and the references cited therein for standarddosing and scheduling. These therapies can be used unchanged, or alteredas needed for a particular patient, in combination with one or morebinding polypeptides of the current disclosure as described herein.

Additional regimens that are useful in the context of the presentdisclosure include use of single alkylating agents such ascyclophosphamide or chlorambucil, or combinations such as CVP(cyclophosphamide, vincristine and prednisone), CHOP (CVP anddoxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone andprocarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD(CHOP plus methotrexate, bleomycin and leucovorin), ProMACE-MOPP(prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide andleucovorin plus standard MOPP), ProMACE-CytaBOM (prednisone,doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin,vincristine, methotrexate and leucovorin) and MACOP-B (methotrexate,doxorubicin, cyclophosphamide, vincristine, fixed dose prednisone,bleomycin and leucovorin). Those skilled in the art will readily be ableto determine standard dosages and scheduling for each of these regimens.CHOP has also been combined with bleomycin, methotrexate, procarbazine,nitrogen mustard, cytosine arabinoside and etoposide. Other compatiblechemotherapeutic agents include, but are not limited to,2-Chlorodeoxyadenosine (2-CDA), 2′-deoxycoformycin and fludarabine.

For patients with intermediate- and high-grade NHL, who fail to achieveremission or relapse, salvage therapy is used. Salvage therapies employdrugs such as cytosine arabinoside, carboplatin, cisplatin, etoposideand ifosfamide given alone or in combination. In relapsed or aggressiveforms of certain neoplastic disorders the following protocols are oftenused: IMVP-16 (ifosfamide, methotrexate and etoposide), MIME(methyl-gag, ifosfamide, methotrexate and etoposide), DHAP(dexamethasone, high dose cytarabine and cisplatin), ESHAP (etoposide,methylpredisolone, HD cytarabine, cisplatin), CEPP(B) (cyclophosphamide,etoposide, procarbazine, prednisone and bleomycin) and CAMP (lomustine,mitoxantrone, cytarabine and prednisone) each with well-known dosingrates and schedules.

The amount of chemotherapeutic agent to be used in combination with themodified antibodies of the current disclosure may vary by subject or maybe administered according to what is known in the art. See for example,Bruce A Chabner et al, Antineoplastic Agents, in GOODMAN & GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 (Joel G. Hardman et al,eds., 9th ed. 1996).

As previously discussed, the binding polypeptides of the presentdisclosure, immunoreactive fragments or recombinants thereof may beadministered in a pharmaceutically effective amount for the in vivotreatment of mammalian disorders. In this regard, it will be appreciatedthat the disclosed binding polypeptides will be formulated to facilitateadministration and promote stability of the active agent.

Pharmaceutical compositions in accordance with the present disclosuretypically include a pharmaceutically acceptable, non-toxic, sterilecarrier such as physiological saline, nontoxic buffers, preservativesand the like. For the purposes of the instant application, apharmaceutically effective amount of the modified binding polypeptide,immunoreactive fragment or recombinant thereof, conjugated orunconjugated to a therapeutic agent, shall be held to mean an amountsufficient to achieve effective binding to an antigen and to achieve abenefit, e.g., to ameliorate symptoms of a disease or disorder or todetect a substance or a cell. In the case of tumor cells, the modifiedbinding polypeptide will typically be capable of interacting withselected immunoreactive antigens on neoplastic or immunoreactive cellsand provide for an increase in the death of those cells. Of course, thepharmaceutical compositions of the present disclosure may beadministered in single or multiple doses to provide for apharmaceutically effective amount of the modified binding polypeptide.

In keeping with the scope of the present disclosure, the bindingpolypeptides of the disclosure may be administered to a human or otheranimal in accordance with the aforementioned methods of treatment in anamount sufficient to produce a therapeutic or prophylactic effect. Thebinding polypeptides of the disclosure can be administered to such humanor other animal in a conventional dosage form prepared by combining theantibody of the disclosure with a conventional pharmaceuticallyacceptable carrier or diluent according to known techniques. It will berecognized by one of skill in the art that the form and character of thepharmaceutically acceptable carrier or diluent is dictated by the amountof active ingredient with which it is to be combined, the route ofadministration and other well-known variables. Those skilled in the artwill further appreciate that a cocktail comprising one or more speciesof binding polypeptides described in the current disclosure may prove tobe particularly effective.

XI. Expression of Binding Polypeptides

In one aspect, the invention provides polynucleotides encoding thebinding polypeptides disclosed herein. A method of making a bindingpolypeptide comprising expressing these polynucleotides are alsoprovided.

Polynucleotides encoding the binding polypeptides disclosed herein aretypically inserted in an expression vector for introduction into hostcells that may be used to produce the desired quantity of the claimedantibodies, or fragments thereof. Accordingly, in certain aspects, theinvention provides expression vectors comprising polynucleotidesdisclosed herein and host cells comprising these vectors andpolynucleotides.

The term “vector” or “expression vector” is used herein for the purposesof the specification and claims, to mean vectors used in accordance withthe present invention as a vehicle for introducing into and expressing adesired gene in a cell. As known to those skilled in the art, suchvectors may easily be selected from the group consisting of plasmids,phages, viruses and retroviruses. In general, vectors compatible withthe instant invention will comprise a selection marker, appropriaterestriction sites to facilitate cloning of the desired gene and theability to enter and/or replicate in eukaryotic or prokaryotic cells.

Numerous expression vector systems may be employed for the purposes ofthis invention. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV), or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals. In some embodiments the clonedvariable region genes are inserted into an expression vector along withthe heavy and light chain constant region genes (e.g., human constantregion genes) syntheticized as discussed above.

In other embodiments, the binding polypeptides may be expressed usingpolycistronic constructs. In such expression systems, multiple geneproducts of interest such as heavy and light chains of antibodies may beproduced from a single polycistronic construct. These systemsadvantageously use an internal ribosome entry site (IRES) to providerelatively high levels of polypeptides in eukaryotic host cells.Compatible IRES sequences are disclosed in U.S. Pat. No. 6,193,980 whichis incorporated by reference herein. Those skilled in the art willappreciate that such expression systems may be used to effectivelyproduce the full range of polypeptides disclosed in the instantapplication.

More generally, once a vector or DNA sequence encoding an antibody, orfragment thereof, has been prepared, the expression vector may beintroduced into an appropriate host cell. That is, the host cells may betransformed. Introduction of the plasmid into the host cell can beaccomplished by various techniques well known to those of skill in theart. These include, but are not limited to, transfection (includingelectrophoresis and electroporation), protoplast fusion, calciumphosphate precipitation, cell fusion with enveloped DNA, microinjection,and infection with intact virus. See, Ridgway, A. A. G. “MammalianExpression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez andDenhardt, Eds. (Butterworths, Boston, Mass. 1988). Plasmid introductioninto the host can be by electroporation. The transformed cells are grownunder conditions appropriate to the production of the light chains andheavy chains, and assayed for heavy and/or light chain proteinsynthesis. Exemplary assay techniques include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), orflourescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

As used herein, the term “transformation” shall be used in a broad senseto refer to the introduction of DNA into a recipient host cell thatchanges the genotype and consequently results in a change in therecipient cell.

Along those same lines, “host cells” refers to cells that have beentransformed with vectors constructed using recombinant DNA techniquesand encoding at least one heterologous gene. In descriptions ofprocesses for isolation of polypeptides from recombinant hosts, theterms “cell” and “cell culture” are used interchangeably to denote thesource of antibody unless it is clearly specified otherwise. In otherwords, recovery of polypeptide from the “cells” may mean either fromspun down whole cells, or from the cell culture containing both themedium and the suspended cells.

In one embodiment, the host cell line used for antibody expression is ofmammalian origin; those skilled in the art can determine particular hostcell lines which are best suited for the desired gene product to beexpressed therein. Exemplary host cell lines include, but are notlimited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus),HELA (human cervical carcinoma), CVI (monkey kidney line), COS (aderivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line),SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI(human lymphocyte), 293 (human kidney). In one embodiment, the cell lineprovides for altered glycosylation, e.g., afucosylation, of theantibodyexpressed therefrom (e.g., PER.C6® (Crucell) or FUT8-knock-outCHO cell lines (Potelligent® Cells) (Biowa, Princeton, N.J.)). In oneembodiment NS0 cells may be used. CHO cells are particularly useful.Host cell lines are typically available from commercial services, theAmerican Tissue Culture Collection or from published literature.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography.

Genes encoding the binding polypeptides featured in the invention canalso be expressed non-mammalian cells such as bacteria or yeast or plantcells. In this regard it will be appreciated that various unicellularnon-mammalian microorganisms such as bacteria can also be transformed;i.e. those capable of being grown in cultures or fermentation. Bacteria,which are susceptible to transformation, include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the polypeptides can become part of inclusionbodies. The polypeptides must be isolated, purified and then assembledinto functional molecules.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available. For expression in Saccharomyces, the plasmidYRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsmanet al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) iscommonly used. This plasmid already contains the TRP1 gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, Genetics, 85:12 (1977)). The presence of the trp1 lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents ofSequence Listing, figures and all references, patents, and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

Example 1. Chemoenzyme Synthesis of CMP-Sialic Acid or CMP-Sialic AcidDerivatives at C5

N-acetyl mannosamine or a derivative thereof can be treated with sialicacid aldolase to form sialic acid or sialic acid derivatives. Subsequenttreatment of the sialic acid or sialic acid derivative with CTP in thepresence of CMP-sialic acid synthetase would result in the creation ofCMP-sialic acid or a CMP-sialic acid derivative (FIG. 1).

CMP-sialic acid derivatives that could be created through thechemoenzyme synthesis outlined in FIG. 1 include, but are not limitedto, the C5 CMP-sialic acid derivatives of FIG. 2. FIG. 2 also showsCMP-sialic acid derivatives at C7 and C8. The CMP-sialic acidderivatives can be used as substrates to transfer the sialic acidderivatives to antibodies through in vitro sialylation for subsequentconjugation.

Example 2. Different Chemistries for Conjugation Through Sialic AcidDerivatives

FIGS. 3A-E are depictions of different chemical reactions of the instantinvention, wherein the circles in combination with the reactive moietiesto which they are bonded represent sialic acid derivative-conjugatedbinding polypeptides. The stars represent targeting or effectormoieties. FIG. 3A is a schematic showing the reacting of a sialic acidderivative-conjugated binding polypeptide comprising a terminal aldehydewith an aminooxy effector moiety, e.g., a drug or a glycan, to form animine FIG. 3B is a schematic showing the reacting of a sialic acidderivative-conjugated binding polypeptide comprising a terminal ketogroup with an aminooxy effector moiety, e.g., a drug or a glycan, toform an imine FIG. 3C is a schematic showing the reacting of a sialicacid derivative-conjugated binding polypeptide comprising a terminalaldehyde or keto with an effector moiety comprising a hydrazine to forma hydrazone, which is a type of imine FIG. 3D is a schematic showing thereacting of a sialic acid derivative-conjugated binding polypeptidecomprising a terminal azide with an effector moiety, e.g., a drug or aglycan, comprising an alkyne or bound to a moiety comprising an alkyne(here, DBCO) to form a triazole. FIG. 3E is a schematic showing thereacting of a sialic acid derivative-conjugated binding polypeptidecomprising a terminal thiol with an effector moiety, e.g., a drug or aglycan, comprising a maleimide to form a thioester bond.

FIG. 4 depicts an effector moiety conjugated binding polypeptideaccording to the methods of the instant invention. The effector moietyconjugated binding polypeptide can be formed by (a) reacting a sialicacid derivative with a glycan of a binding polypeptide to form a sialicacid derivative-conjugated binding polypeptide; and (b) reacting thesialic acid derivative-conjugated binding polypeptide with an effectormoiety to form the effector moiety conjugated binding polypeptide,wherein an imine bond is formed, and wherein neither the bindingpolypeptide nor the sialic acid derivative-conjugated bindingpolypeptide are treated with an oxidizing agent. FIG. 4 depicts theformation of an oxime, a type of imine.

FIG. 5 depicts an effector moiety conjugated binding polypeptideaccording to the methods of the instant invention. The effector moietyconjugated binding polypeptide can be formed by (a) reacting a sialicacid derivative comprising a terminal reactive moiety at the C5 positionwith a glycan of a binding polypeptide to form a sialic acidderivative-conjugated binding polypeptide; and (b) reacting the sialicacid derivative-conjugated binding polypeptide with an effector moietyto form the effector moiety conjugated binding polypeptide using clickchemistry. FIG. 5 depicts the formation of a triazole ring.

Example 3. Design, Preparation, and Characterization of 2C3 Anti-CD-52Hyperglycosylation Antibody Mutants

Multiple hyperglycosylation mutations were designed in the heavy chainof the anti-CD-52 antibody, 2C3, for the purpose of adding a bulky groupto an interaction interface (e.g., the FcRn binding site to modulateantibody pharmacokinetics), for modulating antibody effector function bychanging its interaction with FcγRs, or to introduce a novelcross-linking site subsequence chemical modification for effector moietyconjugation, including but not limited to, drugs, toxins, cytotoxicagents, and radionucleotides. The hyperglycosylated 2C3 mutants are setforth in Table 3.

TABLE 3 Hyperglycosylated 2C3 anti-CD-52 mutants Mutation DesiredBenefit Applications A114N Glycosylation at 1) Control Asn-Ser-Thr 2)Effector moiety conjugation Y436T Glycosylation at 1) Transplant andother Asn434 Inhibition of indications which need FcRn binding shorthalf-life Y436S Glycosylation at 1) Transplant and other Asn434Inhibition of indications which need FcRn binding short half-life S440NGlycosylation at 1) Control Asn-Leu-Ser 2) Effector moiety conjugationS442N Glycosylation at 1) Control Asn-Leu-Ser 2) Effector moietyconjugation Add NGT to C- Glycosylation 1) Control terminal 2) Effectormoiety conjugation S298N/Y300S Glycosylation at 1) Reduce effectorAsn298 function Reduced effector 2) Effector moiety function conjugation3A. Creation of 2C3 Anti-CD-52 Antibody Hyperglycosylation Mutants

The A114N mutation, designated based upon the Kabat numbering system,was introduced into the CH1 domain of 2C3 by mutagenic PCR. To createthe full-length antibody, the VH domain plus the mutated A114N residuewas inserted by ligation independent cloning (LIC) into thepENTR-LIC-IgG1 vector encoding antibody CH domains 1-3. All othermutations were introduced on pENTR-LIC-IgG1 by site-directed mutagenesiswith a QuikChange site-directed mutagenesis kit (Agilent Technologies,Inc., Santa Clara, Calif., USA). The WT 2C3 VH was cloned into mutatedvectors by LIC. Full-length mutants were cloned into thepCEP4(A-E+I)Dest expression vector by Gateway cloning. Fc mutations weredesignated based on the EU numbering system. Mutations were confirmed byDNA sequencing Amino acid sequences of the WT 2C3 heavy and light chainsand the mutated 2C3 heavy chains are set forth in Table 4. Mutated aminoacids are highlighted in gray and the consensus glycosylation targetsites created by the mutation are underlined.

TABLE 4 Amino acid sequences of 2C3 anti-CD-52 antibodies SEQ ID NO NameAmino Acid Sequence 1 Anti- DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTY CD-52LNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSG WTTDFTLKISRVEAEDVGVYYCVQGTHLHTFGQGTRL lightEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP chainREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 Anti-VQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN CD-52WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR WTFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW heavyGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG chainCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3 Anti- CD-52 A114N heavy chainEVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW

CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN CD-52WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR Y436SFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW heavyGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG chainCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC

5 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN CD-52WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR S440N FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW heavyGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG chainCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC

6 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN CD-52WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR S442NFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW heavyGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG chainCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC

7 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN CD-52WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR NGTFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW heavyGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG chainCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC

8 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN CD-52WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR S298N/FTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW Y300SGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG heavyCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG chainLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

The mutants and WT control were transfected into HEK293A-EBNA cells in a6-well plate format. As shown in FIGS. 14A and B, the expression levelwas found to be ˜0.1 μg/ml, as analyzed by SDS-PAGE and Western blot.Expression of mutants in conditioned media was also measured by proteinA capture on Biacore. Concentration was determined using thedissociation response 6 minutes after injection into immobilized ProteinA. CHO-produced WT 2C3 serially diluted in media from 90 μg/mL to 1.5ng/mL was used as a standard curve. Concentrations were calculated downto ˜0.2 μg/mL by a calibration curve using a 4-parameter fit. As shownin FIG. 14B, relative expressions levels were low and generallycorresponded with the Western blot results.

3B. Verification of Hyperglycosylation

To determine whether additional glycosylation sites were introduced bymutation, 2C3 mutant and wild type proteins were treated with theuniversal deglycosylating enzyme PNGase F and protein samples wereanalyzed by SDS-PAGE and Western blot. As shown in FIG. 15, only theA114N mutant had an increased apparent molecular weight, indicating thepresence of an additional N-linked carbohydrate.

Small scale antibody preparations were produced to purify the 2C3mutants for further verification of glycosylation site introduction. Asshown in FIG. 16, it was confirmed by SDS-PAGE that only the A114Nmutant had additional glycosylation sites introduced.

3C. Binding Properties of 2C3 Anti-CD-52 Mutants

Biacore was used to compare the binding properties of the purifiedproteins. Mouse and SEC-purified human FcRn-HPC4 were immobilized on aCM5 chip via amine coupling. Each antibody was diluted to 200, 50, and10 nM and injected over the immobilized Fc receptors. Campath,CHO-produced WT 2C3, and DEPC-treated Campath were included as positiveand negative controls. As shown in FIG. 18, the Y436S mutant displayedabout a 2-fold decrease in binding to human FcRn. Interestingly, bindingof this mutant to mouse FcRn was not affected. None of the other 2C3mutations had any considerable effect on human or mouse FcRn binding.

Biacore was used to compare the antigen binding properties of thepurified proteins using the CD-52 peptide 741 Biacore binding assay.CD-52 peptide 741 and control peptide 777 were immobilized to a CM5chip. Antibodies were serially diluted 2-fold from 60 to 0.2 nM inHBS-EP and injected in duplicate for 3 min followed by a 5 mindissociation in buffer at a 50 μL/min flow-rate. GLD52 lot 17200-084 wasincluded as a control. The surface was regenerated with 1 pulse of 40 mMHCl. A 1:1 binding model was used to fit the 7.5 to 0.2 nM curves. Asshown in FIG. 21, the A114N mutant had a slightly lower CD-52 bindingaffinity while the NGT mutant had a slightly higher affinity than therest of the mutants in this assay. The CD-52 peptide 741 Biacore bindingassay was repeated with protein purified from larger scale prep. Asshown in FIG. 22, the A114N mutant exhibited CD-52 peptide binding thatwas comparable to WT 2C3.

3D. Charge characterization of the A114N mutant

Isoelectric focusing (IEF) was performed to characterize the charge ofthe 2C3 mutants. Purified protein was run on immobilized pH gradient(pH3-10) acrylamide (IPG) gels. As shown in FIG. 23A, A114N was found tohave more negative charges, likely due to sialic acid residues. IntactMS data confirmed the complex structure with sialic acids on the A114Nmutant. In contrast, the WT 2C3 was shown to have G0F and G1F as thedominant glycosylation species (FIGS. 23C and 23D, respectively).

Example 4. Preparation of Hyperglycosylation Mutants in Several AntibodyBackbones

In addition to the 2C3 anti-CD-52 antibody, the A114N mutation wasengineered in several other antibody backbones to confirm that theunique hyperglycosylation site could be introduced into unrelated heavychain variable domain sequences. The hyperglycosylated anti-TEM1,anti-FAP, and anti-Her2 mutants are set forth in Table 5.

TABLE 5 A114N and/or S298N mutants designed in several unrelatedantibody backbones Mutation Antibody Desired benefits Applications A114Nanti-TEM1 Additional glycosylation 1) Control anti-FAP site at the elbowhinge 2) Aminooxy toxin anti-Her2 of heavy chain for site- conjugationvia exposed specific carbohydrate- sialic acid or galactose mediatedconjugation group (SAM or GAM) S298N/ anti-Her2 Switch theglycosylation 1) Aminooxy toxin T299A/ from Asn297 to an conjugation viaexposed Y300S engineered Asn298. Expect sialic acid or galactose (NNASsolvent exposed and group (SAM or GAM) (“NNAS” complex carbohydrates at2) Reduced effector disclosed S298N, offering conjugation function asSEQ site and means to remove ID NO: effector function 40)) A114N/anti-Her2 Potential for increased 1) Control NNAS conjugation yield withtwo 2) Aminooxy toxin (“NNAS” conjugation sites conjugation via exposedas SEQ sialic acid or galactose ID NO: group (SAM or GAM) 40)4A. Creation of Anti-TEM1 and Anti-FAP Antibody HyperglycosylationMutants

The A114N mutation, designated based upon the Kabat numbering system,was introduced into the CH1 domain of anti-TEM1 and anti-FAP bymutagenic PCR. To create the full-length antibody, the mutated VH plusresidue 114 was inserted by ligation independent cloning (LIC) into thepENTR-LIC-IgG1 vector encoding antibody CH domains 1-3. Full-lengthmutants were then cloned into the pCEP4(A-E+I)Dest expression vector byGateway cloning. Mutations were confirmed by DNA sequencing Amino acidsequences of the anti-TEM1 wild type and mutated heavy and light chainsare set forth in Table 6. Mutated amino acids are highlighted in grayand the consensus glycosylation target sites created by the mutation areunderlined.

TABLE 6 Amino acid sequences of anti-TEM1 and anti-FAP antibodies SEQ IDNO Name Amino Acid Sequence  9 Anti- EIVLTQSPGTLSLSPGERATLSCRASQSVSSSTEM1 YLAWYQQKPGQAPRLLIYGASSRATGIPDRFS WTGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSP light WTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSchain  GTASVVCLLNNFYPREAKVQWKVDNALQSGNS (cloneQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV #187) YACEVTHQGLSSPVTKSFNRGEC 10 Anti-QVQLQESAPGLVKPSETLSLTCTVSGGSIRSYYWS TEM1WIRQPPGKGLEYIGYIYYTGSAIYNPSLQSRVTIS WTVDTSKNQFSLKLNSVTAADTAVYYCAREGVRGASG heavyYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSK chainSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH (cloneTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN #187)HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11 Anti-QVQLQESAPGLVKPSETLSLTCTVSGGSIRSYYW TEM1SWIRQPPGKGLEYIGYIYYTGSAIYNPSLQSRVT A114NISVDTSKNQFSLKLNSVTAADTAVYYCAREGVRG

LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

The mutants and wild type control were transfected into HEK293A-EBNAcells in a triple flask format and purified on HiTrap protein A columns(GE Healthcare Biosciences, Pittsburgh, Pa., USA). As analyzed by A280on a NanoDrop spectrophotometer, the expression of anti-FAP A114N andanti-FAP A114C was about 3 μg/ml and about 1 μg/ml, respectively. Theexpression of anti-TEM1 A114N was about 0.04 μg/ml.

4B. Verification of Hyperglycosylation

To confirm that the additional glycosylation site was introduced intothe A114N mutants, purified protein from the A114N mutants was analyzedon reducing SDS-PAGE along with wild-type control protein. Oneadditional glycosylation site would add 2000-3000 Daltons to themolecular weight of the heavy chain. As shown in FIG. 25, SDS-PAGEindicated that the anti-FAP and anti-TEM1 A114N mutant heavy chain bandshad increased apparent molecular weight, consistent with successfulintroduction of an additional glycosylation site to both antibodies.

4C. Creation of Anti-Her2 Antibody Hyperglycosylation Mutants

The Her-2 A114N, Her-2 A114N/NNAS (“NNAS” disclosed as SEQ ID NO: 40),and WT Her-2 antibodies were created by ligation independent cloning.The VH domain of Herceptin was synthesized and PCR-amplified with twoLIC-compatible sets of primers, either WT or bearing the A114N mutation.To obtain a full-length antibody, amplified VH inserts (WT or A114N)were cloned into two pENTR vectors encoding CH 1-3 domains,pENTR-LIC-IgG1 WT and pENTR-LIC-IgG1 NNAS (“NNAS” disclosed as SEQ IDNO: 40), resulting in three full-length mutants (A114N, NNAS (“NNAS”disclosed as SEQ ID NO: 40), A114N/NNAS (“NNAS” disclosed as SEQ ID NO:40)) and WT control as entry clones on pENTR. These mutants were clonedinto the pCEP4(A-E+I)Dest expression vector, by Gateway cloning.Mutations were confirmed by DNA sequencing. Amino acid sequences of theanti-Her-2 wild type and mutated heavy and light chains are set forth inTable 7. Mutated amino acids are highlighted in gray and the consensusglycosylation target sites created by the mutation are underlined.

TABLE 7 Amino acid sequences of anti-Her-2 antibodies SEQ ID NO NameAmino Acid Sequence 12 Anti- DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW Her-2YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGT WTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKV lightEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF chainYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 13 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW Her-2VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS WTADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY heavyAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG chainGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW Her-2VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS A114NADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY heavy

chain GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW Her2VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS NNASADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY (″NNAS″AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG disclosedGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA as SEQ  VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK ID NO:PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV 40)FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF heavy

chain VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 16 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW Her2VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS A114N/ADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY NNAS

(″NNAS″ GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA disclosed VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH as SEQ KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS ID NO:VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV 40)

heavy  TVLHQDWLNGKEYKCKVSNKALPAPIEKTISK chainAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK4D. Expression of the A114N Anti-Her2 Antibody Hyperglycosylation Mutant

The A114N anti-Her2 and wild type constructs were transfected withLipofectamine-2000 (2.5:1 ratio of reagent to DNA) and XtremeGene HP(3:1 ratio of reagent to DNA) into HEK293A-EBNA cells in 12 tripleflasks. Octet measurement of aliquots from day 3 conditioned media (CM)showed that protein expression was consistent across 6 flasks for bothLipofectamine-2000 and XtremeGene HP. As shown in Table 8, the overalltransfection efficiency was about 30% higher with XtremeGene HP.Conditioned media collected on day 3 was pooled together for bothtransfection conditions and purified by protein A column. Octetmeasurement showed 1.8 ug/ml antibody in the serum-containing mock mediaversus 0 ug/ml in no serum mock media.

TABLE 8 A114N anti-Her2 hyperglycosylation mutant expressionLipofectamine- XtremeGene 2000 HP Purified protein Concentration 1.723.18 from protein A (mg/ml) column Volume (ml) 3.5 3.5 Total protein(mg) 6.02 11.13 Buffer-exchanged Concentration 15.59 16.86 protein(mg/ml) Volume (ml) 0.2 0.36 Total protein (mg) 3.1 6.07 % Recovery 51.854.5

Conditioned media from Day 6 was collected and purified separately foreach transfection condition. Both eluates were buffer-exchangedseparately into PBS, pH 7.2, and concentrated ˜15-fold using Amicon-4(50 kD cut-off) columns Day 6 CM showed higher expression level comparedto Day 3 CM. As shown in Table 8, a total of 3 mg of Herceptin A114N15.59 mg/ml (from Lipofectamine transfection) and 6 mg of HerceptinA114N 16.86 mg/ml (from XtremeGene HP transfection) was produced fromday 6 conditioned media for additional downstream applications, such asantibody-drug conjugation.

4E. SDS-PAGE and HIC Analysis of the A114N Anti-Her2 Mutant

Prior to conjugation, purified A114N Herceptin was characterized bySDS-PAGE and HIC (hydrophobic interaction chromatography). As shown inFIG. 26, the quality of purified A114N Herceptin was determined to besuitable for further downstream applications.

4F. Conjugation to Engineered Glycosylation

It was demonstrated that: a) a glycosylation site was introduced atKabat position 114 site on anti-TEM1; b) the A114N mutant hadhyperglycosylation on the heavy chain by reducing SDS-PAGE; and c) theA114N hyperglycosylated mutant had complex carbohydrate structure byintact LC/MS, including terminal sialic acids and galactose, which areideal for SAM and GAM conjugation. To confirm that the engineeredglycosylation site was suitable for conjugation, anti-TEM1 A114N wasconjugated with a 5 kDa PEG via aminooxy chemistry. As shown in FIG. 27,PEG was successfully conjugated to anti-TEM1 A114N through an aminooxylinkage. This mutant was also successfully prepared on the anti-FAP andanti-CD-52 2C3 backbones (not shown). These data demonstrate that theglycosylation site at N114 is useful for conjugation of effectormoieties.

Example 5. Generation of S298N/Y300S Fc Mutants

Engineered Fc variants was designed and generated in which a newglycosylation site was introduced at EU position Ser 298, next to thenaturally-occurring Asn297 site. The glycosylation at Asn297 was eithermaintained or ablated by mutation. Mutations and desired glycosylationresults are set forth in Table 9.

TABLE 9 Glycosylation states of various antibody variants # MutationDesired Glycosylation State Applications A N297Q No glycosylation (agly)Agly Control B T299A No glycosylation (agly) Agly Control, unknowneffector function C N297Q/S298N/ No glycosylation at 297 Reducedeffector Y300S (NSY) but engineered function; Conjugation glycosylationsite at 298 via exposed sialic acid or galactose groups. D S298N/T299A/No glycosylation at 297 Reduced effector Y300S (STY) but engineeredfunction; Conjugation glycosylation site at 298 via exposed sialic acidor galactose groups. E S298N/Y300S Two potential glycosylation Reducedeffector (SY) sites at 297 & 298; function; Conjugation Alterations inglycosylation via exposed sialic pattern. acid or galactose groups. FWild-type 297 control5A. Creation of H66 αβ-TCR Antibody Altered Glycosylation Variants

Mutations were made on the heavy chain of αβ T-cell receptor antibodyclone #66 by Quikchange using a pENTR_LIC_IgG1 template. The VH domainof HEBE1 Δab IgG1 #66 was amplified with LIC primers before being clonedinto mutated or wild type pENTR_LIC_IgG1 by LIC to create full-lengthmutant or wild-type antibodies. The subcloning was verified withDraIII/XhoI double digest, producing an approximately 1250 bp-sizedinsert in the successful clones. Those full-length mutants were thencloned into an expression vector, pCEP4(A-E+I)Dest, via Gateway cloning.The mutations were confirmed by DNA sequencing Amino acid sequences ofthe WT H66 anti-αβTCR heavy and light chains and the mutated H66 heavychains are set forth in Table 10. Mutated amino acids are highlighted ingray and the consensus glycosylation target sites created by themutation are underlined.

TABLE 10 Amino acid sequences of H66 anti-αβTCR  antibodies SEQ  ID NOName Amino Acid Sequence 23 Anti-  EIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQαβTCR KPGQAPRRLIYDTSKLASGVPARFSGSGSGTSYTLTI clone  SSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKRTVAA H66PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK light VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY chainEKHKVYACEVTHQGLSSPVTKSFNRGEC* 24 Anti- EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW αβTCRVRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR clone  DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF H66VYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA heavy LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG chainLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM  ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 25 Anti- EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW αβTCRVRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR clone  DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF H66VYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA S298N/LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG Y300S LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV heavy EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM chainISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK

SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 26 Anti- EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW αβTCRVRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR clone  DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF H66VYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA S298N/LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG T299A/LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV Y300SEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM heavy ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK chain

SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 27 Anti- EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW αβTCRVRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR clone   DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF H66VYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA N297Q/LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG S298N/LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV Y300SEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM heavy ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK chain

SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK*

The mutant, wild-type, and two aglycosylated control (HEBE1 Agly IgG4and HEBE1 Δab IgG1 in pCEP4) constructs were transfected intoHEK293A-EBNA cells in triple-flasks for expression. Proteins werepurified from 160 ml of conditioned media (CM) with 1 ml HiTrap proteinA columns (GE) using a multi-channel peristaltic pump. Five microgramsof each resulting supernatant were analyzed on 4-20% Tris-Glycinereducing and non-reducing SDS-PAGE gels (see FIG. 7). The heavy chainsof the aglycosylated mutants (N297Q, T299A, and Agly controls), havemigrated further (arrowhead), consistent with the loss of the glycans inthese antibodies. The heavy chains of the engineered glycosylatedantibodies (NSY, STY, SY, Δab, and wt control, arrows), however, migratesimilarly to the wild-type control. This result is consistent with theexistence of an engineered glycosylation site at EU position 298.SEC-HPLC analysis indicated that all mutants are expressed as monomers.

5B. Glycosylation Analysis by LC-MS

The engineered H66 IgG1 Fc variants were partially reduced with 20 mMDTT at 37° C. for 30 min. The samples were then analyzed by capillaryLC/MS on an Agilent 1100 capillary HPLC system coupled with a QSTAR qqTOF hybrid system (Applied Biosystems). A Bayesian proteinreconstruction with baseline correction and computer modeling in AnalystQS 1.1 (Applied Bisoystem) was used for data analysis. In theS298N/T299A/Y300S H66 antibody mutant, one glycosylation site wasobserved at amino acid 298 with bi-antennary and tri-antennarycomplex-type glycans detected as the major species alongside G0F, G1Fand G2F (see FIG. 39). This altered glycosylation profile is consistentwhich shifted glycosylation at N298 instead of the wild-typeglycosylation site at N297.

5C. Binding Properties of αβTCR Antibody Mutants to Human FcγRIIIa andFcγRI Using Biacore

Biacore was used to assess binding to recombinant human FcγRIIIa (V158 &F158) and FcγRI. All four flowcells of a CM5 chip were immobilized withanti-HPC4 antibody via the standard amine coupling procedure provided byBiacore. The anti-HPC4 antibody was diluted to 50 μg/mL in 10 mM sodiumacetate pH 5.0 for the coupling reaction and injected for 25 min at 5μL/min Approximately 12,000 RU of antibody was immobilized to the chipsurface. Recombinant human FcγRIIIa-V158 and FcγRIIIa-F158 were dilutedto 0.6 μg/mL in binding buffer (HBS-P with 1 mM CaCl₂) and injected toflowcells 2 and 4, respectively, for 3 min at 5 μL/min to capture300-400 RU receptor on the anti-HPC4 chip. In order to distinguishbetween the low binders, three times more rhFcγRIIIa was captured on theanti-HPC4 surface than usually used in this assay. Flowcells 1 and 3were used as reference controls. Each antibody was diluted to 200 nM inbinding buffer and injected over all four flowcells for 4 min, followedby 5 min dissociation in buffer. The surfaces were regenerated with 10mM EDTA in HBSA-EP buffer for 3 min at 20 μL/min. The results of theseexperiments are shown in FIG. 8.

Biacore was also used to compare the FcγRI binding. Anti-tetra Hisantibody was buffer exchanged into 10 mM sodium acetate pH 4.0 using aZeba Desalting column and diluted to 25 μg/mL in the acetate buffer foramine coupling. Two flowcells of a CM5 chip were immobilized with ˜9000RU of the anti-Tetra-His antibody after 20 min injection at 5 μL/min. Asin the previous experiment, ten times more FcγRI was captured to theanti-tetra-His surface in order to compare samples with weak binding.Recombinant human FcγRI was diluted 10 μg/mL in HBSA-EP binding bufferand injected to flowcell 2 for 1 min at 5 μL/min to capture ˜1000 RUreceptor to the anti-tetra-His chip. A single concentration of antibody,100 nM, was injected for 3 min at 30 μL/min over the captured receptorand control surface. Subsequently, dissociation was monitored for threeminutes. The surface was then regenerated with two 30 second injectionsof 10 mM glycine pH 2.5 at 20 μL/min. The results of these experimentsare shown in FIG. 9.

These results demonstrate a striking decrease in binding of theglycoengineered mutants to FcγRIIIa or FcγRI. H66 S298N/T299A/Y300S inparticular has almost completely abolished binding to both receptors.This mutant was chosen for more detailed analysis.

5D. Stability Characterization Using Circular Dichroism (CD)

The stability of the S298N/T299A/Y300S antibody mutant was monitored bya Far-UV CD thermo melting experiment in which the CD signal at 216 nmand 222 nm was monitored as increasing temperature lead to the unfoldingof the antibody (denaturation).

Temperature was controlled by a thermoelectric peltier (Jasco modelAWC100) and was increased at a rate of 1° C./min from 25-89° C. The CDspectra were collected on a Jasco 815 spectrophotometer at a proteinconcentration of approximately 0.5 mg/mL in PBS buffer in a quartzcuvette (Hellma, Inc) with a path length of 10 mm. The scanning speedwas 50 nm/min and a data pitch of 0.5 nm. A bandwidth of 2.5 nm was usedwith a sensitivity setting of medium. The CD signal and HT voltage werecollected from 210-260 nm with data intervals of 0.5 nm and attemperature intervals of 1° C. and four replicate scans were performedfor each sample. The results demonstrate that both delta AB H66 and theS298N/T299A/Y300S H66 mutant exhibit similar thermal behaviors and haveapproximately the same onset temperature for degradation (around 63° C.)(FIG. 40), further suggesting that they have comparable stability.

Example 6. Functional Analysis of Fc-Engineered Mutants

Fc-engineered mutants were assessed through a PBMC proliferation assayand a cytokine release assay. In the PBMC proliferation assay, humanPBMC were cultured with increasing concentrations of therapeuticantibody for 72 hours, ³H-thymidine was added and cells were harvested18 hours later. For the T cell depletion/Cytokine Release assay, humanPBMC were cultured with increasing concentrations of therapeuticantibody and were analyzed daily for cell counts and viability (Vi-Cell,Beckman Coulter) out to day 7. Cell supernatants were also harvested,stored at −20° C. and analyzed on an 8-plex cytokine panel (Bio-Rad).

Normal donor PBMC were thawed and treated under the following conditions(all in media containing complement): Untreated; BMA031, moIgG2b 10ug/ml; OKT3, moIgG2a 10 ug/ml; H66, huIgG1 deltaAB 10 ug/ml, 1 ug/ml and0.1 ug/ml; H66, huIgG1 S298N/T299A/Y300S 10 ug/ml, 1 ug/ml and 0.1ug/ml.

Cytokines were harvested at day 2 (D2) and day 4 (D4) for BioplexAnalysis (IL2, IL4, IL6, IL8, IL10, GM-CSF, IFNg, TNFa). Cells werestained at D4 for CD4, CD8, CD25 and abTCR expression.

The results, shown in FIGS. 10-13, demonstrate that H66S298N/T299A/Y300S behaved similarly to the H66 deltaAB in all the cellbased assays performed, showing minimal T-cell activation by CD25expression, binding to abTCR (with slightly different kinetics todeltaAB), and minimal cytokine release at both D2 and D4 time points.The S298N/T299A/Y300S mutant thus eliminated effector function aseffectively as the deltaAB mutation.

Example 7. Preparation and Characterization of an Engineered Fc Variantin the Anti-CD52 Antibody Backbone

In addition to the H66 anti-αβTCR antibody, the S298N/Y300S mutation wasalso engineered in an anti-CD52 antibody backbone (clone 2C3). Thismutant was then examined in order to determine whether the observedeffector function modulation seen in the S298N/Y300S H66 anti-αTCRantibody was consistent in another antibody backbone.

7A. Creation of 2C3 Anti-CD52 Antibody Altered Glycosylation Variants

First, S298N/Y300S 2C3 variant DNA was prepared by quick changemutagenesis using pENTR_LIC_IgG1, and WT 2C3 VH was cloned into themutated vector by LIC. Full-length mutants were cloned into the pCEP4(A-E+I)Dest expression vector using Gateway technology. Mutations weresubsequently confirmed by DNA sequencing and the sequences are set forthin Table 11. The mutants were then transfected into HEK293A-EBNA cellsin a 6-well plate format and the protein was purified from conditionedmedia. Anti-CD52 2C3 wild-type antibody was produced in parallel as acontrol. The expression level was found to be 0.1 μg/mL using SD-PAGEand Western blot analyses (FIG. 15A). Expression of mutants in neatconditioned media was also measured by protein A capture on Biacore.Concentration was determined using the dissociation response after asix-minute injection to immobilized protein A. CHO-produced WT 2C3serially diluted in media from 90 μg/mL to 1.5 ng/mL was used as astandard curve. Concentrations were calculated within approximately 0.2μg/mL by a calibration curve using a 4-parameter fit. Relativeexpression levels were low and generally agree with the Western blotdata (FIG. 15B).

TABLE 11 Anti-CD52 clone 2C3 antibody sequences SEQ   ID NO NameAmino Acid Sequence 28 Anti- DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNCD-52 WLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTL 2C3 KISRVEAEDVGVYYCVQGTHLHTFGQGTRLEIKRTVAAP WTSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN lightALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV chain YACEVTHQGLSSPVTKSFNRGEC*29 Anti- EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNWVRQ CD-52APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSK 2C3 NSLYLQMNSLKTEDTAVYYCTPVDFWGQGTTVTVSSAST WTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN heavySGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY chainICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK* 30 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNWVRQ CD-52APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSK 2C3NSLYLQMNSLKTEDTAVYYCTPVDFWGQGTTVTVSSAST S298N/KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWN Y300SSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY heavy ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG chainPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN

GKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK*7B. Glycosylation Analysis Using PNGaseF

To evaluate the additional glycosylation sites introduced by themutation, the enriched S298N/Y300S mutant was de-glycosylated withPNGase F. It did not demonstrate any apparent change in molecularweight, which indicates that no additional carbohydrate was present(FIG. 15). Small scale preparations were performed in order to purifythese mutants for further characterization and the results reconfirmedthat there was not an additional carbohydrate present on the S298N/Y300Smutant (FIG. 16).

7C. Binding Properties of 2C3 Anti-CD52 Antibody Mutants to HumanFcγRIIIa Using Biacore

Biacore was also used to characterize the antigen-binding, FcγRIII, andbinding properties of the purified antibodies (see FIGS. 17A-C, 18, and19A and B). The S298N/Y300S 2C3 variant bound to the CD52 peptidetightly and the binding sensorgram was undistinguishable from thewild-type control, demonstrating that this mutation does not affect itsantigen binding (FIG. 17A).

To assay for Fc effector function, FcγRIII receptor (Val158) was used inbinding studies. The mutant and wild-type control antibody were dilutedto 200 nM and injected to HPC4-tag captured FcγRIIIa. FcγRIII bindingwas almost undetectable for the S298N/Y300S mutant, which indicated aloss of effector function by this variant (FIG. 17B and FIG. 19A). Tofurther assay for Fc effector function, the FcγRIII receptor (Phe158)was also used in binding studies. The mutant and wild-type controlantibodies were diluted to 200 nM and injected to HPC4-tag capturedFcγRIIIa. FcγRIII binding was almost undetectable for the S298N/Y300Smutant, which indicates a loss of effector function with the Phe158variant (FIG. 19B). Finally, Biacore was used to compare the FcRnbinding properties of the purified proteins. Mouse and SEC-purifiedhuman FcRn-HPC4 were immobilized to a CM5 chip via amine coupling. Eachantibody was diluted to 200, 50, and 10 nM and injected over thereceptors. Campath, CHO-produced WT 2C3, and DEPC-treated Campath wereincluded as positive and negative controls. These data show that themutant binds to both human and murine FcRn receptor with the sameaffinity as the wild-type antibody control and that it likely has noalterations in its circulation half-life or other pharmacokineticproperties. (see FIG. 17C, FIG. 18). Accordingly, the S298N/Y300Smutation is applicable to antibodies in general, to reduce or eliminateundesired Fc effector function, for example through engagement of humanFcγ receptors.

Example 8. Circulating Immune Complex Detection in the S298N/Y300SMutant

Circulating immune complex detection was also investigated using a C1qbinding assay for the S298N/Y300S mutant and WT control. High bindingCostar 96-well plates were coated overnight at 4° C. with 100 μl of2-fold serially diluted 2C3 Abs at concentrations ranging from 10-0.001μg/ml in coating buffer (0.1M NaCHO₃ pH 9.2). ELISA analysis showed thatC1q binding is reduced for the S298N/Y300S mutant compared to WT (FIG.20A). The binding of anti-Fab Ab to the coated 2C3 Abs confirmedequivalent coating of the wells (FIG. 20B).

Example 9. Separation and Analysis of S298N/Y300S Mutant UsingIsoelectric Focusing

A pH 3-10 Isoelectric Focusing (IEF) gel was run to characterize theS298N/Y300S mutants. 5298/Y300S was found to have more negative charges,and therefore, likely more sialic acid molecules (FIG. 23A). Both theS298N/Y300S mutant and WT 2C3 were shown by intact MS to have G0F andG1F as the dominant glycosylation species (FIGS. 23B and D,respectively).

Example 10. Antigen Binding Affinity of S298N/Y300S

Biacore was used to compare the antigen binding affinity of WT anti-CD522C3 Ab and the S298N/Y300S mutant that had been prepared and purifiedfrom both smaller (FIG. 21) and larger (FIG. 22) scale expressions. CM5chips immobilized with CD52 peptide 741 and control peptide 777 wereobtained. Antibodies were serially diluted 2-fold from 60 to 0.2 nM inHBSA-EP and were then injected over the chip surface for 3 min followedby a 5 min dissociation in buffer at a flow rate of 50 μl/min. Thesurface was then regenerated with a pulse of 40 mM HCl. These analyseswere performed in duplicate and demonstrate that the S298N/Y300S mutantand WT 2C3 antibodies show comparable CD52 peptide binding.

A media screening platform was designed to test functional bindingproperties prior to purification in order to screen antibodies createdduring small scale transfections. These tests were performed using Octet(FIG. 24A) to determine concentration and used Protein A biosensors anda GLD52 standard curve. Samples were diluted to 7.5 and 2 nM in HBSA-Epfor a CD52 binding comparison using Biacore (FIG. 24B). The results ofthe peptide binding assay showed that both the S298N/Y300S mutant andthe WT 2C3 antibodies have comparable CD52 peptide binding. Furthermore,these analyses demonstrate that Octet and Biacore work well to predictantigen binding by antibodies from small scale transfections.

Example 11. Preparation of S298N/Y300S, S298N/T299A/Y300S, andN297Q/S298N/Y300S Altered Glycosylation Mutants in Additional AntibodyBackbones

In addition to the anti-αβ-TCR antibody and 2C3 anti-CD-52 antibody, the5298/Y300S, S298N/T299A/Y300S, and N297Q/S298N/Y300S mutations wereengineered in other antibody backbones to confirm that the additionaltandem glycosylation site could be introduced into unrelated heavy chainvariable domain sequences. The alternatively glycosylated anti-CD-5212G6 and anti-Her2 mutants are set forth in Tables 12 and 13.

TABLE 12 Anti-CD52 clone 12G6 antibody sequences SEQ   ID NO NameAmino Acid Sequence 31 Anti- DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNWVCD-52 LQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISR 12G6 VEAEDVGVYYCVQGSHFHTFGQGTKLEIKRTVAAPSVFIFP WTPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS lightQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ chain GLSSPVTKSFNRGEC 32 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ CD-52APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN 12G6 SLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG WTPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL heavyTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH chainKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 33 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ CD-52APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN 12G6SLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG S298N/PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL Y300STSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH heavy KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP chainKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 34 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ CD-52APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN 12G6 SLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG S298N/PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL T299A/TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH Y300SKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP heavy KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV chain

SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 35 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ CD-52APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN 12G6 SLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG N297Q/PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL S298N/TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH Y300SKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP heavy KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV chain

SNKALPAPIEKTTSKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*

TABLE 13 Anti-Her2 antibody sequences SEQ   ID NO NameAmino Acid Sequence 36 Anti- DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPHer2 GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQP WTEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP lightSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ chainESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC* 37 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA Her2PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY WTLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS heavyASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS chainWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK* 38 Anti-EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA Her2PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY S298N/LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS T299A/SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS Y300SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY heavy ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS chainVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV

YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK*

Example 12. Generation of Altered Antibodies Containing Reactive GlycanMoieties

In order to generate antibodies containing glycan moieties capable ofreacting with derivatized effector moieties, an anti-HER antibody wasfirst glycosylated in vitro using glycosyltransferase and relevant sugarnucleotide donors. For example, to introduce the sialic acid residues,donor antibodies were first galactosylated with β-galactosyltransferase,followed with sialylation with α2,6-sialyltransferase according to themethods of Kaneko et al. (Kaneko, Y., Nimmerjahn, F., and Ravetch, J. V.(2006) Anti-inflammatory activity of immunoglobulin G resulting from Fcsialylation. Science 313, 670-3). The reaction was performed in aone-pot synthesis step using β-galactosyltransferase (50 mU/mg, Sigma)and α2,6-sialyltranafrease (5 ug/mg, R&D system) with donor sugarnucleotide substrates, UDP-galactose (10 mM) and CMP-sialic acid (10 mM)in 50 mM MES buffer (pH 6.5) containing 5 mM MnCl₂. The reaction mixturecontaining 5 mg/ml anti-HER2 antibody was incubated for 48 hours at 37°C. The sialylation was verified using MALDI-TOF MS analysis ofpermethylated glycans released from the antibody with PNGase F, sialicacid content analysis using Dionex HPLC and lectin blotting with SNA, alectin specific for α2,6-sialic acid.

MALDI-TOF analysis of glycans released by PNGase F treatment of thesialylated anti-HER2 antibody indicated that native glycans had beencompletely remodeled with a mainly monosialylated biantennary structure,A1F (FIG. 32A) together with small amount of disialylated species.Treatment of the antibody with higher amounts of α2,6-sialyltransferaseproduced more homogenous populations of the A1F glycoform, suggestingthat either the enzyme activity or glycan localization may prevent fullsialylation. Sialic acid content was determined to be ˜2 mol per mol ofantibody, which is consistent with A1F glycan as the major glycoformspecies (FIG. 32B). Lectin blotting with a SAN lectin, Sambucus nigraagglutinin specific for α2,6-linked sialic acid, confirmed that thesialic acid was present in an α2,6-linkage configuration (FIG. 32C).

In conclusion, although the native protein glycans are somewhatheterogeneous, remodeling through galactosyl and sialyltransferasesyields a nearly homogeneous antibody with monosialylated but fullygalactosylated biantennary glycans (A1F). The introduction of only ˜1sialic acid on the two galactose acceptors on each branched glycan maybe due to limited accessibility of one of the galactoses from glycanswhich are often buried in the antibody or non-covalent interactions ofthe glycans with the protein surface.

Example 13. Alternate Method: Oxidation of Altered Antibodies ContainingReactive Glycan Moieties

Oxidation of sialylated anti-HER2 antibody with various concentrationsof periodate (0.25 to 2 mM) was investigated. The sialylated antibodywas first buffer-exchanged into 25 mM Tris-HCl (pH 7.5) containing 5 mMEDTA followed by buffer exchange with PBS buffer. The buffered antibodymixture was then applied to protein A Sepharose column pre-equilibratedwith PBS buffer. After the column was washed with 15 column volumes ofPBS, 15 column volumes of PBS containing 5 mM EDTA, and 30 columnvolumes of PBS, it was then eluted with 25 mM citrate phosphate buffer(pH 2.9). The eluates were immediately neutralized with dibasicphosphate buffer and the antibody concentrated using Amicon ultra fromMillipore. Following purification, the sialylated anti-HER2 antibodythen was oxidized with sodium periodate (Sigma) in 100 mM sodium acetatebuffer (pH 5.6) on ice in the dark for 30 minutes, and the reactionquenched with 3% glycerol on ice for 15 minutes. The product wasdesalted and exchanged into 100 mM sodium acetate (pH 5.6) by 5 roundsof ultrafiltration over 50 kDa Amicons. FIG. 33A shows sialic acidcontent analysis of sialylated antibody titrated with various amounts ofperiodate. Complete oxidation of the sialic acid residues was achievedat a periodate concentration above 0.5 mM. A periodate concentration aslow as 0.5 mM was enough to fully oxidize the introduced sialic acid.Accordingly, a 1 mM concentration of periodate was chosen for oxidationof sialylated antibody for drug conjugation.

Oxidation can have adverse effects on the integrity of an antibody. Forexample, the oxidation of methionine residues, including Met-252 andMet-428, located in Fc CH3 region, close to FcRn binding site is knownto affect FcRn binding which is critical for prolonging antibody serumhalf-life (Wang, W., et al. (2011) Impact of methionine oxidation inhuman IgG1 Fc on serum half-life of monoclonal antibodies. Mol Immunol48, 860-6). Accordingly, to examine the potential side effects ofperiodate oxidation on methionine residues (e.g., Met-252) critical forFcRn interaction, the oxidation state of the sialylated antibody wasdetermined by LC/MS analysis of a trypsin peptide digest. This analysisrevealed ˜30% oxidation of Met-252 and <10% oxidation of Met-428 aftertreatment of the sialylated trastuzumab with 1 mM periodate. Todetermine the impact of this degree of methionine oxidation on FcRnbinding, the FcRn binding kinetics for each antibody was evaluated usingsurface plasmon resonance (BIACORE). This analysis revealed thatoxidation state correlated with a minor loss in FcRn binding (12% and26% reduction to mouse and human FcRn, see FIGS. 33B and 33Crespectively). Notably, a ˜25% reduction in the Ka for human FcRn hasbeen reported to have no effect on the serum half-life in a human FcRntransgenic mouse, since a single intact FcRn site on each antibody issufficient to provide functionality and the PK advantage (Wang et al.,Id).

In summary, these data indicate that the introduction ofperiodate-sensitive sialic acid residues by sialyltransferase treatmentpermits the use of much lower concentrations of periodate, resulting inlowered side effects on antibody-FcRn interactions and antibodyintegrity as assessed by aggregation (≤1%).

The galactose in a hyperglycosylated antibody mutant can also beoxidized specifically using galactose oxidase to generate an aldehydegroup for conjugation. To confirm this approach, an A114N anti-TEM1antibody was concentrated to 13-20 mg/ml and then treated with 20 mU/mgsialidase in PBS for 6 hours at 37° C. The desialated product was thenoxidized with galactose oxidase (“GAO”), first with 5 ug GAO/mg proteinovernight at 37° C. followed by addition of 2 ug GAO/mg protein andincubation for an additional 5 hours. Sodium acetate was added to adjustthe pH to 5.6 (0.1 v/v, pH5.6), and DMSO was added to achieve a finalreaction concentration of 16%, were added prior to conjugation. Thehyperglycosylation mutant A114N anti-HER antibody (15 mg/ml) wassimilarly desialylated with sialidase (20 mU/mg) and oxidized with 5 ugGAO per mg protein in a single reaction overnight at 37° C.

Example 14. Synthesis of Reactive Effector Moieties

In order to facilitate conjugation with the aldehyde-derivatizedantibody glycoforms, candidate drug effector moieties (e.g., MomomethylAuristatin E (MMAE) and Dolastatin 10 (Do110)) were derivatized withaminooxy-cystamide to contain functional groups (e.g., aminooxy-cys)specifically reactive with the aldehyde.

Briefly, to generate aminooxy-cystamide as a starting material,S-Trityl-L-cysteinamide (362 mg, 1 mmol) was added to a 3 mL of a DMFsolution of t-BOC-aminooxyacetic acid N-hydroxysuccinimide ester (289mg, 1 mmol). The reaction was complete after 3 h as evident from HPLCanalysis. The reaction mixture was subsequently diluted with 30 ml ofdichloromethane and was washed with 0.1 M sodium bicarbonate solution(2×20 mL), water (2×20 mL), and brine (2×20 mL). The solution was driedover anhydrous sodium sulfate, filtered and concentrated to dryness. Tothis dried residue was added 3 mL of TFA followed by 150 μL oftriethylsilane. The resulting solution was precipitated from t-butylmethyl ether and the process repeated three times. After filtration, theresidue was dried under reduced pressure yielding 205 mg of an off whitesolid (67% yield). The compound was used for next step without furtherpurification.

To generate aminooxy-derivatized MMAE (Aminooxy-Cys-MC-VC-PABC-MMAE),30.1 mg of aminooxy-cystamide (0.098 mmol, 2 eq.) was combined with 64.6mg of MC-VC-PABC-MMAE (0.049 mmol), and 100 μL of triethylamine in 3 mLof DMF. The resulting reaction mixture was stirred at room temperaturefor 15 minutes, by which time reaction was complete according to HPLCanalysis. The compound was purified by preparative HPLC yielding 45 mg(62%) of the desired product as an off-white solid. Reversed-phase HPLCanalysis suggested the purity of the compound to be >96%. ESI calcd forC73H116N140185 (MH)⁺1509.8501. found, m/z 1509.8469.

To generate aminooxy-derivatized Do110(Aminooxy-Cys-MC-VC-PABC-PEG8-Do110), 7.4 mg (0.024 mmol, 3 eq.) ofaminooxy-cystamide, 12 mg (0.008 mmol) of MC-VC-PABC-PEG8-Do110 and 30μL triethylamine were combined in in 3 mL of DMF. The reaction wascomplete within 15 minutes according to HPLC analysis. Preparative HPLCpurification resulted in 6.2 mg (46%) of the desired product as anoff-white solid. Reversed-phase HPLC analysis suggests the purity of thecompound to be >96%. ESI calcd for C80H124N1601952 (MH)⁺1678.0664.found, m/z 1678.0613.

Example 15. Sialic Acid-Mediated (SAM) Conjugation of Reactive EffectorMoieties

Following desalting, drug-linkers of Example 13 were combined with theoxidized, sialylated antibodies of Example 12 with 75% DMSO (0.167 v/v)at a concentration of 25 mM to achieve a 24:1 molar ratio of drug-linkerto antibody and a final antibody concentration at 5 mg/ml. The mixturewas incubated overnight at room temperature. The unincorporateddrug-linkers and any free drugs were scavenged using BioBeads. Theproduct was buffer-exchanged into Histidine-Tween buffer using PD-10columns and sterile filtered. The endotoxin levels were determined andless than 0.1 EU/mg ADC was achieved for in vivo study.

FIG. 34A-C shows a hydrophobic interaction chromatograph (HIC) ofdifferent sialylated antibodies (anti FAP B11 and G11 and the anti-HER2antibody of Example 13) glycoconjugated to AO-MMAE. Sialylated HER2antibody was also conjugated with the drug-linker,AO-Cys-MC-VC-PABC-PEGS-Do110 (FIG. 34D). This analysis reveals thatthere are mainly one or two drug conjugates per antibody with adrug-to-antibody ratio (DAR) ranging from 1.3-1.9. The increasedretention time of the Do110 glycoconjugate (FIG. 34D) as compared to theMMAE glycoconjugate (FIG. 34C) is likely due to the greaterhydrophobicity of Do110.

LC-MS analysis was also conducted with an anti-HER antibody conjugatedwith two different drug-linkers (AO-MMAE or AO-PEG8-Do110) at 30 mgscale. This analysis showed similar DAR values of 1.7 and 1.5 followingconjugation, which is comparable to HIC analysis. Size-exclusionchromatograpy (SEC) showed very low levels (1%) of aggregates in theseconjugates.

Example 16. Galactose-Mediated (GAM) Conjugation of Reactive EffectorMoieties

The galactose aldehyde generated with galactose oxidase on the A114NantiTEM1 hyperglycosylation mutant antibody as described in Example 13was conjugated with 24 molar excess of aminooxy-MC-VC-PABC-MMAEdrug-linker over antibody by overnight incubation at 25° C., yielding aADC conjugate with a DAR of 1.72.

To the galactose oxidase-treated anti-HER antibody prepared as describedin Example 13, one tenth reaction volume of 1M sodium acetate, pH5.6,was added to adjust the pH to 5.6 and DMSO was added to make the finalconcentration of 14% before adding 24 eq aminooxy MC-VC-PABC-MMAE druglinker. The reactions were incubated for overnight at room temperature.Free drug and drug-linker were scavenged with Biobeads and the productbuffer exchanged by SEC (65% yield). The product conjugate was analyzedby HIC. As shown in FIG. 35, AO-MMAE had been conjugated to ˜60% of themolecules.

Example 17. In Vitro ADC Cell Proliferation Assays

The in vitro activity of the anti-HER and anti-FAP glycoconjugatemolecules were also compared with corresponding thiol conjugatescontaining the same drug moiety linked via thiol linkages to hingeregion cysteines of the same donor antibody. The thiol conjugatescontained approximately twice the number of drugs per antibody (DAR)than the glycoconjugates. Thiol-based conjugation was performed asdescribed by Stefano et al (Methods in Molecular Biology 2013, inpress). Her2+ SK-BR-3 and Her2− MDA-MB-231 cell lines were then employedto evaluate the relative efficacy of each ADC. The results of thisanalysis are presented in Table 15 below

TABLE 15 EC₅₀ comparison of glycoconjugates and thiol conjugates DAREC₅₀ (ng/ml) Anti-HER-MC-VC-PABC-MMAE 3.8* 2.3 (Thiol MMAE)Anti-HER-AO-Cys-MC-VC-PABC-MMAE 1.7* 4.7 (Glyco MMAE)Anti-HER-MC-VC-PABC-PEG8-Dol10 3.9* 0.45 (Thiol Dol10)Anti-HER-AO-Cys-MC-VC-PABC-PEG8- 1.5* 0.97 Dol10 (Glyco Dol10) Anti FAPB11-MC-VC-PABC-MMAE  3.3** 382.4 (Thiol MMAE), CHO + FAP Anti FAPB11-AO-Cys-MC-VC-PABC-  1.5** 682.4 MMAE (Glyco MMAE), CHO + FAP Note:*DAR determined by LC-MS; **DAR determined by HIC

FIG. 36A-D shows a comparison of in vitro potency of anti-HERglycoconjugate and its counterpart thiol conjugate. Cell viability wasdetermined following 72 hr exposure of the conjugates to Her2 antigenexpressing (SK-BR-3) cells (FIGS. 36A and C) or non-expressing(MDA-MB-231) cells (FIGS. 36B and D). The ADCs contained either MMAE orPEGS-Do110 linked to the glycans (“glyco”) or by conventional chemistryto hinge region cysteines (“thiol”). As shown in FIGS. 36A and C,˜2-fold lower EC₅₀ was observed for the thiol conjugates compared to theglycoconjugates, which is consistent with 2-fold higher DAR in theformer than the latter. No toxicity was observed with the Her2-cell linewith any antibody up to 100 ug/ml.

Similar trends were also observed in the cell proliferation for ADCprepared with antibodies against a tumor antigen (FAP) which is highlyexpressed by reactive stromal fibroblasts in epithelial cancersincluding colon, pancreatic and breast cancer (Teicher, B. A. (2009)Antibody-drug conjugate targets. Curr Cancer Drug Targets 9, 982-1004).These conjugates were again prepared by conjugating either aminooxy MMAEdrug-linker or maleimido MMAE drug-linker to glycans or a thiol group.Cell proliferation assays of these conjugates showed that EC₅₀ of thethiol conjugate had ˜100-fold higher potency on the CHO cellstransfected with human FAP than the same cells lacking FAP expression asdepicted in FIG. 37, which shows a comparison of in vitro potency ofanti FAP B11 glycoconjugate and thiol conjugate. Cell viability wasdetermined following exposure of the conjugates to CHO cells transfectedwith or without FAP antigen. The ADCs contained MMAE linked to theglycans (“glyco”) or by conventional chemistry to hinge region cysteines(“thiol”). Note that the ˜2-fold lower EC50 for the thiol compared tothe glycoconjugates is consistent with the relative amounts of drugdelivered per antibody assuming similar efficiencies for target bindingand internalization in antigen expressing CHO cells. In parallel, aglycoconjugate of anti FAP (B11) ADC with a DAR of 1.5 as describedpreviously was assayed and showed an ˜2-fold higher EC₅₀ than comparatorthiol conjugate (DAR 3.3).

As shown in FIG. 41, similar trends were observed in the cellproliferation assay for ADC prepared with the anti-HER antibody bearingthe A114N hyperglycosylation mutation and AO-MMAE as described inExample 16, when assayed on SK-BR-3 expressing cells or MDA-MB-231cells. The A114N glycoconjugate clearly shows enhanced cell toxicityagainst the Her2 expressing cell line over the non-expressing line. Therelative toxicity compared to the SialT glycoconjugate prepared with thesame antibody is consistent with the lower drug loading of thispreparation.

A cell proliferation assay was also performed for ADC prepared with theanti-TEM1 antibody bearing the A114N hyperglycosylation mutation andAO-MMAE prepared as described in Example 16. Higher toxicity wasobserved with the TEM1-expressing cells lines SJSA-1 and A673 comparedto the non-expressing MDA-MB-231 line. The level of toxicity comparedwith a conventional thiol conjugate with the same antibody was inkeeping with the drug loading (DAR) of this preparation.

A673- A673-DMEM- MDA- SJSA-1 RPMI RPMI MB-231 IC50 IC50 IC50 IC50antiTEM1 A114N- 3 μg/ml 3.2 μg/ml 2.2 μg/ml 40 μg/ml AO-MC-VC-PABC- MMAEantiTEM1-MC-VC- 4 μg/ml   1 μg/ml 0.9 μg/ml 20 μg/ml PABC-MMAE

In summary, the site-specific conjugation of the drugs through theglycans with cleavable linkers produces ADCs with toxicities and invitro efficacy that are equivalent to conventional thiol-basedconjugates, as demonstrated using different antibodies and differentdrug-linkers. Moreover, below 2 mM periodate, the level of drugconjugation correlates with the reduction of sialic acid. Increasingperiodate concentration above 2 mM produces little benefit, as expectedfrom the complete conversion of sialic acid to the oxidized form.However, under all conditions, the number of drugs per antibody wasslightly lower than the sialic acid content, indicating that some of theoxidized sialic acids may similarly not be available for coupling,either because of being buried or otherwise due to steric hindrancearising from the bulk of the drug-linker.

Example 18. In Vivo Characterization of Antibody Drug Conjugates

Efficacy of anti-HER glycoconjugates were also evaluated in a Her2+tumor cell xenograft mode and compared with thiol conjugate comparatorshaving ˜2-fold higher DAR. Beige/SCID mice were implanted with SK-OV-3Her2+ tumor cells which were allowed to establish tumors of ˜150 mm³prior to initiation of treatment. ADCs at 3 or 10 mg/kg doses wereinjected through tail vein on days 38, 45, 52 and 59. There were ˜10mice per group. The tumor volume of mice in different group was measuredand their survival was recorded. The survival curve was plotted based onKaplan-Meier method.

FIG. 38A-D shows a comparison of in vivo efficacy of the anti-HERglycoconjugates and thiol conjugates in a Her2+ tumor cell xenograftmodel. Beige/SCID mice implanted with SK-OV-3 Her2+ tumor cells weredosed with MMAE (FIGS. 38A and B) and PEGS-Do110 (FIGS. 38C and D)containing glycoconjugates or a thiol conjugate comparators with ˜2-foldhigher DAR. The tumor growth kinetics of the MMAE conjugates is shown inFIG. 38A. In this case, the glycoconjugate showed a significantly higherefficacy than the naked antibody alone (black) but less than a thiolconjugate comparator having a ˜2-fold higher DAR (green). The MMAEglycoconjugate showed significant tumor regression and a ˜20 day delayin tumor growth (FIG. 38A) and ˜2-fold increase in survival time fromfirst dose (FIG. 38B). The thiol MMAE conjugate showed near-completetumor suppression at the same dose of ADC (10 mg/kg).

The in vivo efficacy of a PEGS-Do110 glycoconjugate (“Glyco Do110”) anda thiol conjugate comparator with ˜2-fold higher DAR (“Thiol Do110”) wasalso determined in the same Her2+ tumor cell xenograft model. Bothconjugates showed lower efficacy than MMAE conjugates as describedpreviously. However, the aminooxy-PEGS-Do110 glycoconjugate (“GlycoDo110”) at 10 mg/kg showed a 15-day delay in tumor growth (FIG. 38C) and˜20 day (1.7-fold) increase in survival time following firstadministration (FIG. 38D). The thiol conjugate was more efficacious atthe same dose, showing a 2-fold increase in survival. At a lower dose (3mg/kg), the thiol conjugate showed a lesser efficacy than theglycoconjugate at 10 mg/kg. This dose corresponds to 80 umol PEGS-Do110drug per kg dose, compared to 110 umol PEG8-Do110 drug per kg dose forthe glycoconjugate.

These data demonstrate that site-specific conjugation of drugs ontosialic acid of antibody glycans yields molecules with comparable potencyas ADCs generated via thiol-based chemistry. The somewhat lower in vivoefficacy likely stems from the fewer number of drugs which are carriedby each antibody into the tumor cells by the internalization of eachantibody-bound antigen. Although we have not compared theseglycoconjugates with thiol conjugates of the same DAR, the efficacyobserved at different doses of the two ADCs representing comparablelevels of administered drug shows that the glycoconjugates havecomparable intrinsic efficacy as their thiol counterparts, indicating nodeleterious effect of conjugation at this site. Moreover, a 10 mg/kgdose of the Do110 glycoconjugate which introduced only 28% more drugprovided a 2-fold increase in survival over the thiol conjugate (at 3mg/kg), suggesting these conjugates may even provide superior efficaciesat the same DAR. Given the apparent limitation in sialic acidincorporation at native glycans, higher drug loading could be achievedby a number of different strategies including the use of branched druglinkers or the introduction of additional glycosylation sites and usingthe same method.

Example 19. Conjugation of Targeting Moieties

FIG. 42 demonstrates the overall scheme for the conjugation of targetingmoieties to existing carbohydrates or engineered glycosylation sites.This conjugation can be performed through the attachment of neoglycans,glycopeptides, or other targeting moieties to oxidized sialylatedantibodies (FIGS. 43 and 44). Moieties suitable for conjugation mayinclude those containing aminooxy linkers (FIGS. 45 and 46).

Example 20. Conjugation Through Sialic Acid in Native Fc Glycans

Mannose-6-P hexamannose aminooxy was conjugated to either a polyclonalantibody or monoclonal antibody specifically targeting a Man-6-Preceptor. The SDS-PAGE and Maldi-TOF analyses of the conjugation of theanti-Man-6-P-receptor rabbit polyclonal antibody with Man-6-Phexamannose aminooxy is shown in FIG. 47. FIG. 48 depicts the results ofsurface plasmon resonance experiments used to assess the binding ofcontrol and Man-6-P hexamannose conjugated anti-Man-6-P-receptor rabbitpolyclonal IgG antibodies to M6P receptor. In vitro analyses of thisconjugated antibody demonstrates increased uptake into both HepG2 (Homosapiens liver hepatocellular carcinoma) and RAW (Mus musculus murineleukemia) cell lines (FIG. 49). Cultures were stained withanti-rabbit-Alexa 488 antibody counterstained with DAPI.

Antibodies conjugated with M6P or lactose aminooxy moieties were furthertested through SDS-PAGE and lectin blotting and compared withunconjugated antibodies (FIG. 50). MALDI-TOF intact protein analyses ofthe control and conjugated antibodies demonstrate that the conjugateshave approximately two glycan moieties per antibody, while controlantibodies have none (FIG. 51).

Example 21. Conjugation Through Sialic Acid to Hinge Cysteine Residuesin Antibody

Mannose-6-P hexamannose maleimide was conjugated to either a polyclonalantibody or monoclonal antibody specifically targeting a Man-6-Preceptor.

The conjugation of a polyclonal antibody with Man-6-P hexamannosemaleimide through hinge cysteines was examined through SDS-PAGE, lectinblotting, and M6P quantitation (to determine the number of glycansconjugated per antibody) (FIG. 52). Conjugation of a polyclonal antibodywith lactose maleimide was also examined through the use of SDS-PAGE andgalactose quantitation of the control antibody, conjugated antibody, andfiltrate are shown in FIG. 53. Little increased aggregation was observedin hinge cysteine-conjugated polyclonal antibodies by size exclusionchromatography (SEC) (FIG. 55).

The conjugation of a monoclonal antibody with Man-6-P hexamannosemaleimide through hinge cysteines was also examined through SDS-PAGE andglycan quantitation (to determine the number of glycans conjugated perantibody) (FIG. 54). Little increased aggregation was observed in hingecysteine-conjugated polyclonal antibodies by size exclusionchromatography (SEC) (FIG. 56).

The conjugation of bisM6P hexasaccharide to polyclonal and monoclonalantibodies through native Fc glycans or hinge disulfides was alsoexamined through native PAGE (FIG. 60).

Example 22. Preparation of Sialylated Monoclonal Antibody andConjugation to a Trigalactosylated Glycopeptide or Glycopeptide

A mouse monoclonal antibody mutant with an STY mutation (NNAS (“NNAS”disclosed as SEQ ID NO: 40)) was modified with sialidase andgalactosyltransferase for making mainly native trigalactosylated glycans(2 glycans per antibody). The same mutant was also sialylated withsialyltransferase and conjugated with a glycopeptide using SAM approach.The sialic acid content of the enzyme modified antibodies was examined(FIG. 57). Further, MALDI-TOF analysis of the glycans released fromcontrol and desialylated/galactosylated (FIG. 58) NNAS (“NNAS” disclosedas SEQ ID NO: 40) as well as the glycans released from control andsialylated (FIG. 59) NNAS (“NNAS” disclosed as SEQ ID NO: 40) wereexamined SDS-PAGE (4-12% NuPAGE) and lectin blotting of enzyme modifiedand conjugated NNAS (“NNAS” disclosed as SEQ ID NO: 40) are shown inFIG. 61. Terminal galactose quantitation was also measured for thecontrol NNAS (“NNAS” disclosed as SEQ ID NO: 40) antibody,desialylated/galactosylated NNAS (“NNAS” disclosed as SEQ ID NO: 40)antibody, and conjugated NNAS (“NNAS” disclosed as SEQ ID NO: 40)antibody (FIG. 62).

Example 23. Preparation of α2,3 Sialylated Lactose Maleimide Using aChemoenzyme Approach and Subsequent Conjugation to Non-Immune Rabbit IgGThrough Hinge Disulfides

As carbohydrate-binding proteins (including Siglec proteins) prefermultivalent binding for strong interaction, the monosialylated glycanson a given antibody may not provide enough sialic acid density for otherSiglec proteins. Therefore, a hinge disulfide conjugation approach forintroducing multiple copies of sialylated glycans was investigated. Toproduce sialylated glycans for conjugation, lactose maleimide (5 mg) wassialylated in vitro with α2,3 sialyltransferase from Photobacteriumdamsela in Tris buffer (pH 7.5), for 2 hrs at 37° C. A control glycanwas incubated without sialyltransferase and compared with the originalglycans. MALDI-TOF analysis showed that the incubation of lactosemaleimide without enzyme in Tris buffer (pH 7.5) for 2 hrs at 37° C. didnot change the expected molecular weight of the molecule, suggestingthat the examined condition did not result in maleimide hydrolysis. TheMALDI-TOF and Dionex HPLC analysis of glycans modified with α2,3sialyltransferase indicate the presence of sialyllactose, although notas major peak (data not shown). Therefore, the sialyllactose maleimidewas additionally purified using QAE-sepharose columns and each fractionwas subsequently analyzed using MALDI-TOF and Dionex HPLC. Theseanalyses indicated that sialyllactose maleimide existed as major speciesin the 20 mM NaCl eluate from QAE column (FIG. 63). The amount ofsialylated glycans purified was estimated using sialic acid quantitationanalysis of the samples, indicating a recovery of ˜1.8 mg sialyllactosemaleimide.

Subsequent conjugation of a rabbit polyclonal antibody with thissialyllactose maleimide was tested using thiol chemistry. A rabbit IgGantibody (1 mg) was reduced with TCEP at a 4 molar excess (over theantibody) for 2 hrs at 37° C. before being conjugated to 24 molar excessof sialyllactose for 1 hr at room temperature. The conjugate was thenbuffer-exchanged into PBS for analysis on SDS-PAGE (FIG. 64A). Sialicacid quantitation was also performed using Dionex HPLC (FIG. 64B).Aliquots of control and thiol conjugate were treated with or withoutsialidase (1 U per mg) overnight at 37° C. before supernatants wererecovered through filtration (10 kDa MWCO). The sialic acid content ofthe supernatants was measured and compared to samples treated withoutsialidase. There are approximately 4 α2,3 sialyllactose moieties coupledper antibody.

Example 24. Preparation of α2,6 Sialyllactose Maleimide by SilylatingLactose Maleimide and Conjugation to Hinge Disulfides of a RabbitPolyclonal Antibody Through α2,3- or α2,6-Linkages Resulting in HighSialylation

The conjugation of multiple copies of either α2,3- or α2,6-sialylatedglycans to the hinge disulfides of a rabbit polyclonal antibody wasinvestigated. Since the α2,3 sialyllactose maleimide was successfullyproduced using a chemoenzyme approach (see above, Example 23), a similarmethod was used to produce α2,6 sialyllactose maleimide (minormodifications of the protocol included the use of a differentsialyltransferase). To produce α2,6 sialylated glycan for conjugation,lactose maleimide (˜5 mg) was sialylated in vitro with 0.5 U of abacterial α2,6 sialyltransferase from Photobacterium damsela in Trisbuffer (pH 8) for 1 hr at 37° C. After enzymatic reaction, the productwas applied to a QAE-sepharose column. The column was washed with 10fractions of 1 ml 2 mM Tris (pH 8), 5 fractions of 1 ml of Tris buffercontaining 20 mM NaCl, and 5 fractions of 1 ml Tris buffer containing 70mM NaCl. The aliquots from each fraction were analyzed using Dionex HPLCalongside lactose and α2,6 sialyllactose standards. The oligosaccharideprofiles of the standards and one of the eluted fractions are shown inFIG. 65 (A-D). The fractions containing α2,6 sialyllactose maleimidewere also analyzed and confirmed by MALDI-TOF. The glycan in one of thefractions can be seen in FIG. 66.

The amount of α2,6 sialyllactose maleimide purified was then estimatedusing sialic acid quantitation analysis which indicated a recovery of˜1.5 mg sialyllactose maleimide. Once the glycan was prepared, theconjugation of antibody with either α2,6 sialyllactose maleimide or α2,6sialyllactose maleimide was tested using thiol chemistry. A rabbitpolyclonal IgG antibody (1 mg) was buffer-exchanged and reduced withTCEP at a 4 molar excess (over antibody) for 2 hrs at 37° C. The reducedantibody was then split in half: one portion was conjugated to 24 molarexcess of α2,6 sialyllactose maleimide, and the other to α2,6sialyllactose maleimide for 1 hr at room temperature. The two conjugateswere then buffer-exchanged into PBS before SDS-PAGE analysis (FIG. 67,A) and sialic acid quantitation using Dionex HPLC (FIG. 67, B). Sialicacid quantitation was used to estimate the number of glycan conjugated.Aliquots of control antibody and thiol-conjugated antibody were treatedwith or without sialidase (1 U per mg) overnight at 37° C. beforesupernatants were recovered through filtration (10 kDa MWCO). The sialicacid content of the supernatants was measured and compared to samplestreated without sialidase (control). The analysis demonstrated thatapproximately 7 glycans (either α2,6- or α2,6-sialyllactose glycans)were conjugated to the polyclonal antibody by this method.

Example 25. PEGylation of NNAS (“NNAS” Disclosed as SEQ ID NO: 40) UsingGAM Chemistry

A mouse NNAS (“NNAS” disclosed as SEQ ID NO: 40) (S298N/T299A/Y300S)mutant monoclonal antibody was galactosylated and disialylated,generating a Gal NNAS (“NNAS” disclosed as SEQ ID NO: 40) monoclonalantibody without any protease degradation. This antibody was modifiedwith galactose oxidase (GAO) to generate galactose aldehyde. Thegalactose aldehyde was then conjugated with 2 or 5 kDa of aminooxypolyethylene glycol (PEG). FIG. 68 depicts the characterization ofcontrol and enzyme modified (disalylated/galactosylated) NNAS (“NNAS”disclosed as SEQ ID NO: 40) mutant antibodies using SDS-PAGE and lectinblotting. FIG. 69 depicts the characterization through reducing SDS-PAGEof the PEGylation of a control antibody and Gal NNAS (“NNAS” disclosedas SEQ ID NO: 40) with various amounts of galactose oxidase. Theseresults demonstrate that Gal NNAS (“NNAS” disclosed as SEQ ID NO: 40)can be PEGylated efficiently with significant amounts of mono-, bi-, andtri-PEG conjugated per heavy chain. FIG. 71 depicts the characterizationthrough reducing SDS-PAGE of the PEGylation of a control antibody andGal NNAS (“NNAS” disclosed as SEQ ID NO: 40) with various molar excessof PEG over antibody. Protein Simple scans characterizing the PEGylationthe antibodies demonstrate that approximately 1.5-1.7 PEG moieties areconjugated per heavy chain (or about 3-3.4 PEG per antibody) (FIGS. 70and 72).

Example 26. PEGylation of NNAS (“NNAS” Disclosed as SEQ ID NO: 40) UsingGAM Chemistry

An NNAS (“NNAS” disclosed as SEQ ID NO: 40) antibody was galactosylatedwith 50 mU/mg galactosyltransferase and subsequently desialylated with 1U/mg sialidase in 50 mM MES buffer (pH 6.5). Desialylated fetuin andNNAS (“NNAS” disclosed as SEQ ID NO: 40) as well as galactosylated NNAS(“NNAS” disclosed as SEQ ID NO: 40) were then treated with galactose

oxidase (57 mU/mg)/catalase in the presence or absence of 0.5 mM copperacetate before conjugation with 25 molar excess of 5 kDa aminooxy PEG(FIG. 74, A). In another experiment, galactosylated NNAS (“NNAS”disclosed as SEQ ID NO: 40) was treated with galactose oxidase (57mU/mg)/catalase in the presence of 0, 0.02, 0.1 and 0.5 mM copperacetate before conjugation with 25 molar excess of 5 kDa aminooxy PEG(FIG. 74, B). Antibody oxidized with galactose oxidase in the presenceof copper acetate showed a higher degree of PEGylation than the sameantibody reacted with galactose oxidase in the absence of copperacetate. Significantly higher levels of PEGylation were observed whenthe conjugation was performed in a reaction containing copper sulfate inconcentrations above 0.1 mM.

Example 27. Modification of Wild-Type and Mutant Herceptin UsingSialidase/Galactosyltransferase

Wild-type and mutant (A114N, NNAS (“NNAS” disclosed as SEQ ID NO: 40),and A114N/NNAS) Herceptin antibodies were enzymaticically modified with50 mU/mg galactosyltransferase and subsequently desialylated with 1 U/mgsialidase in 50 mM MES buffer (pH 6.5). The modified antibodies wereanalyzed using SDS-PAGE (reducing and nonreducing), lectin blotting withECL (a plant lectin specific for terminal galactose), and terminalgalactose quantitation using Dionex HPLC analysis of released galactoseby galactosidase (FIG. 75). Enzyme modified antibodies containingapproximately three to nine terminal galactose were obtained with theNNAS (“NNAS” disclosed as SEQ ID NO: 40) and NNAS (“NNAS” disclosed asSEQ ID NO: 40)/A114N double mutants demonstrating a higher level ofterminal galactose than the wild-type and A114N mutant.

Example 28. PEGylation of Wild-Type and Mutant Antibodies Using the SAMConjugation Method

Wild-type and (A114N, NNAS (“NNAS” disclosed as SEQ ID NO: 40), andA114N/NNAS (“NNAS” disclosed as SEQ ID NO: 40)) Herceptin antibodieswere PEGylated using sialic acid-mediated (SAM) conjugation. Theantibodies were subsequently oxidized with 2 mM periodate. After bufferexchange, the oxidized antibodies were PEGylated with 25 molar excess of5 kDa aminooxy PEG. The sialic acid content of the wild-type and mutantantibodies was measured using Dionex HPLC (FIG. 76). The PEGylatedantibodies were then analyzed using reducing and non-reducing SDS-PAGE(FIG. 77). Further, the PEGylation (PAR, number of PEG per antibody) wasestimated by analyzing the scanned gels using ProteinSimple (FIG. 78).The NNAS (“NNAS” disclosed as SEQ ID NO: 40), A114N, and A114N/NNAS(“NNAS” disclosed as SEQ ID NO: 40) mutants all showed higher PAR(2.7-4.6) than wild-type Herceptin antibodies (1.4).

Example 29. Investigation of Uptake of Glycoengineered Antibodies withGalactose Containing Glycan Ligands

A polyclonal antibody was either enzymatically modified withgalactosyltransferase (Gal Transferase), conjugated to lactose aminooxy(Gal-Glc to 297: conjugated to sialic acid in glycans from Asn-297 ofsialylated antibody), or conjugated to lactose maleimide (Gal-Glc toHinge: conjugated to cysteines in hinge disulfides). The control,modified, or conjugated antibodies were then incubated with HepG2 cells(a hepatocyte cell line expressing ASGPR) for 1-2 hrs at 37° C. beforethe uptaken antibodies were measured using Immunofluorescence staining(FIG. 79). The results showed increased HepG2 cell uptake of enzymaticmodified or lactose conjugated antibodies.

Example 30. Conjugation of a Trivalent GalNAc Glycan to Herceptin

Herceptin (anti-Her2) was sialylated and conjugated with a trivalentGalNAc glycan (FIG. 80) for targeting ASGPR using the SAM approach.Subsequently, surface plasmon resonance experiments (Biacore) were usedto assess the binding of these trivalent GalNAc glycan-conjugatedantibodies to ASGPR receptor subunit H1 (FIG. 81).

Example 31. Conjugation of Trivalent GalNAc and Trivalent Galactose to aRecombinant Lysosomal Enzyme

A recombinant lysosomal enzyme was conjugated with either trivalentGalNAc glycan or trivalent galactose containing glycopeptides (FIG. 82)for targeting ASGPR using the SAM conjugation method. Subsequently,surface plasmon resonance experiments (Biacore) were used to assess thebinding of these trivalent GalNAc glycan-conjugated and trivalentgalactose-conjugated enzymes to ASGPR receptor subunit H1 (FIG. 83). Theresults showed strong ASGPR binding of trivalent GalNAc glycanconjugated recombinant lysosomal enzyme.

Example 32. Use of Mannosamine Derivatives, Including ManLev, for InVitro Antibody Sialylation and Conjugation

Mannosamine derivatives, including ManLev, ManNAz, and ManAz, were usedto prepare sialic acid derivatives and then CMP-sialic acid derivativesfor antibody sialylation followed by site-specific conjugation. TheCMP-sialic acid derivatives prepared were characterized using HPAEC-PADand used for in vitro antibody sialylation. Finally, the sialylatedantibodies were PEGylated without periodate oxidation using the SAMapproach.

Sialic acid (0.2 μmol) was titrated with various amounts of CMP-sialicacid synthetase (N. mentingitidis) at 37° C. The generation ofCMP-sialic acid was monitored using HPAEC-PAD as compared to theretention time of CMP-sialic acid standard. The CMP-sialic acidsynthesized versus the amounts of enzyme used was plotted anddemonstrates that generation of CMP-sialic acid is saturated byCMP-sialic acid synthetase at 5 mU per 0.2 μmol (FIG. 84).

ManNAc or ManLev (0.2 μmol) was titrated with various amounts of sialicacid aldolase (E. coli K-12) at 37° C. The generation of sialic acid(from ManNAc) or sialic acid derivative (from ManLev) was monitoredusing HPAEC-PAD as compared to the retention time of sialic acidstandard. The synthesized sialic acid or sialic acid derivative vs theamounts of the enzyme used are shown in FIG. 85 (MacNAc) and FIG. 86(ManLev).

In order to characterize sialic acid derivatives using HPAEC-PAD, theCMP-sialic acid (from ManNAc) or CMP-sialic acid derivative (fromManLev) was first digested with sialidase at 37° C. The released sialicacid or sialic acid derivative was monitored using HPAEC-PAD as comparedto the retention time of sialic acid standard and the identity of sialicacid was also confirmed by disappearance of the sialic acid peak afterperiodate treatment (FIG. 87). Sialic acid derivative (from ManLev) waseluted later than sialic acid.

CMP-sialic acid (from ManNAc) and CMP-sialic acid derivatives (fromManLev, ManNAz, ManAz) were also analyzed directly on HPAEC-PAD withoutsialidase pretreatment. The generation of CMP-sialic acid was comparedto the retention time of CMP-sialic acid standard. The CMP-sialic acidderivatives, produced from ManLev, ManNAz, and ManAz, showed differentretention time compared to CMP-sialic acid standard (FIGS. 88 and 89).

Further, Herceptin was sialylated in vitro using α2,6 sialyltransferaseand CMP-sialic acid derivatives. FIG. 90 is a schematic representationdemonstrating the sialylation of Herceptin using a sialic acidderivative prepared from ManLev. The sialylation was analyzed usingLC-MS of CH₂CH₃ fragments released by IdeS protease. FIG. 91demonstrates the sialylation of Herceptin with the sialic acidderivative prepared from ManLev (with correct mass).

Finally, Herceptin sialylated with sialic acid derivatives prepared fromManLev and ManNAz was PEGylated. FIG. 92 is a schematic representationdemonstrating the PEGylation of Herceptin sialylated with a sialic acidderivative prepared from ManLev. First, the Herceptin was sialylated invitro using α2,6 sialyltransferase and CMP-sialic acid derivativesprepared from ManLev. Subsequently, the sialylated antibodies were mixedwith 5 kDa aminooxy PEG. The sialylated and PEGylated antibodies werethen analyzed using SDS-PAGE under reducing and non-reducing conditions.An SDS-PAGE analysis of sialylated Herceptin PEGylated with a sialicacid derivative prepared from ManLev is seen in FIG. 93. FIG. 94 is aschematic representation demonstrating the sialylation of antibody witha sialic acid derivative prepared from ManNAz. An SDS-PAGE analysis ofPEGylated Herceptin pre-sialylated with a sialic acid derivativeprepared from ManNAz in shown FIG. 95.

We claim:
 1. A method of making an effector moiety conjugated antibodyor antigen-binding fragment thereof comprising the steps of: (a)reacting a cytidine monophosphate-sialic acid (CMP-sialic acid)derivative having the structure:

with a glycan attached to a glycosylation site of an antibody orantigen-binding fragment thereof to form a sialic acidderivative-conjugated antibody having the structure:

or antigen-binding fragment thereof through a sialylation reaction;wherein R₁ is selected from the group consisting of NHC(O)CH₃,NHC(O)CH₂OH, —NHC(O)CH₂CH₂C(O)CH₃, —NHC(O)CH₂N₃, —NHC(O)SH, —OH, and—N₃; and (b) reacting the R₁ group of the sialic acidderivative-conjugated antibody or antigen-binding fragment from step (a)with an effector moiety selected from the group consisting of: a drugmoiety, a cytotoxic agent, a targeting agent, a diagnostic agent, ananti-cancer agent, an anti-inflammatory agent, an anti-cancer agent, ananti-infective agent, and an anesthetic agent, to form the effectormoiety conjugated antibody or antigen-binding fragment thereof usingclick chemistry, wherein step (b) is performed in the absence of copper.2. The method of claim 1, wherein R₁ is N₃.
 3. The method of claim 1,wherein the CMP-sialic acid derivative has the structure of:


4. The method of claim 2, wherein the effector moiety comprises or isbound to a cyclooctyne.
 5. The method of claim 4, wherein thecyclooctyne is an azadibenzocyclooctyne.
 6. The method of claim 4,wherein the cyclooctyne is a monofluorinated cyclooctyne.
 7. The methodof claim 1, wherein the antibody or antigen-binding fragment thereof isan antibody.
 8. The method of claim 1, wherein the antibody comprises anS298N mutant.
 9. The method of claim 1, wherein the antibody comprises aheavy chain of SEQ ID NO: 14 and a light chain of SEQ ID NO:
 12. 10. Themethod of claim 1, wherein step (a) is catalyzed by α2,6sialyltransferase.
 11. The method of claim 1, wherein the effectormoiety comprises a terminal aminooxy moiety or is bound to a moietycomprising an aminooxy derivative.
 12. The method of claim 1, whereinthe effector moiety comprises a poly(ethylene glycol).
 13. The method ofclaim 2, wherein step (b) occurs at ambient temperatures.
 14. The methodof claim 2, wherein the click reaction in step (b) is catalyzed by ametal other than copper.
 15. The method of claim 1, wherein theCMP-sialic acid derivative has the structure of:


16. The method of claim 1, wherein the effector moiety comprises analkyne or is bound to a moiety comprising an alkyne.
 17. The method ofclaim 1, wherein the product of the click reaction in step (b) forms atriazole ring.
 18. The method of claim 4, wherein the cyclooctyne is adifluorinated cyclooctyne.
 19. The method of claim 1, wherein theantibody or antigen-binding fragment thereof comprises an A114N mutantin the CH1 domain.
 20. The method of claim 14, wherein the metal isselected from the group consisting of ruthenium, nickel, palladium,platinum, iron, and a derivative thereof.
 21. The method of claim 1,wherein the effector moiety is selected from the following: